Zinc oxide

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

GLP compliance:

not specified

Remarks:

GLP compliance not specified in publication

Specific details on test material used for the study:

70nm

Analytical monitoring:

no

Vehicle:

no

Test organisms (species):

Daphnia magna

Test type:

static

Water media type:

freshwater

Total exposure duration:

48 h

Test temperature:

20°C

pH:

7.8 +/- 0.2

Reference substance (positive control):

no

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 1.7 - < 9 mg/L

Nominal / measured:

nominal

Conc. based on:

dissolved

Basis for effect:

mobility

Remarks on result:

other: nano ZnO

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 5 - < 16.2 mg/L

Nominal / measured:

nominal

Conc. based on:

dissolved

Basis for effect:

mobility

Remarks on result:

other: bulk ZnO

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 1.4 - < 2.5 mg/L

Nominal / measured:

nominal

Conc. based on:

dissolved

Basis for effect:

mobility

Remarks on result:

other: ZnSO4.7H2O

Reported statistics and error estimates:

One-way analysis of variance (ANOVA) followed by t-testswas used to determine statistical significance of the differences between toxic effects of the compounds in different test media. The differences were considered significant, when p < 0.05.

Test media	NanoZnO	Bulk ZnO	ZnSO4.7H2O
Crustacean Daphniamagna, 48h EC50 (mean± SD, mg metal/l)
AFWa	2.6 ±1.04	7.1±1.1	1.4±0.24
River 1	3.3±1.15	9.5±1.8	1.8±0.32
River 2	9.0 ±0.28	12.0±2.1	2.0±0.19
River 3	1.7±0.27	6.9±0.55	1.6±0.14
River 4	3.5±0.30	>5.0	2.5±0.18
River 5	2.8±0.39	16.2±2.3	1.4± 0.18
River 6	3.4±1.56	10.8±1.4	1.7±0.13

Validity criteria fulfilled:

yes

Conclusions:

Tests done according to standard protocol. Good quality and considered useful for setting the reference value for acute aquatic ecotoxicity

Executive summary:

The D. magna EC50 for nanoZnO, bulk ZnO and ZnSO4 increased only by 1–1.75, 1–2.3 and 0.7–3.6-fold, respectively and in most cases the changes were not statistically significant. These results are in accordance with the data obtained with Zn sensor bacteria showing only small differences in bioavailable Zn ion concentrations in AFWand river waters. Thus, it can be concluded that the toxicity of nano and bulk ZnO in natural waters was due to the solubilised Zn-ions.

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

2004

GLP compliance:

not specified

Remarks:

GLP compliance not specified in publication

Specific details on test material used for the study:

Zinc oxide nanopowders, nominally constituted by ZnO NPs <50 nm-diameter (sZnO) and <100 nm-diameter (bZnO) were purchased from SigmaeAldrich (Milan, Italy): lot number #MKBD9623V and #MKBJ7906V, respectively

Analytical monitoring:

yes

Vehicle:

no

Test organisms (species):

Daphnia magna

Details on test organisms:

The organisms used in this study derived from a single clone of D. magna Straus obtained from the Istituto Superiore di Sanita` (Roma, Italy; courtesy of Dr. Silvia Marchini). D. magna specimen were cultured (30 ind/L) in a commercial mineral water (San Benedetto). Cultured daphnids were fed a suspension of the unicellular green alga Pseudokirchneriella subcapitata (0- to 8-day-old daphnids) and from 8-day-old daphnids: yeast Saccharomyces cerevisiae three times a week. The culture medium was renewed and offspring discarded three times a week. Brood daphnids were renewed every 5/6 weeks and replaced with neonatal organisms

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test temperature:

20.5 +/- 0.5°C

pH:

7.42 and always under 8.8

Conductivity:

415 µs/cm

Nominal and measured concentrations:

Five nZnO suspensions (0.1, 0.33, 1, 3.3 and 10 mg/L) were tested for each NP, together with control batches.
additional exposures with soluble Zn++ were performed at 0.1 mg/L by adding the required mass of ZnSO4.7H2O (Sigma Aldrich, Italy) to the test medium

Details on test conditions:

photoperiod 16h light, 8h dark

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

1.9 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

element (total fraction)

Remarks:

from nano ZnO doped Al

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

2.5 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

element (total fraction)

Remarks:

from nano ZnO

Basis for effect:

mobility

Reported statistics and error estimates:

Immobilization frequency vs. NP concentration was analyzed with Probit analysis and choosing Log transformed concentration data because of the best fit (p < 0.001 for the two tests). Differences in immobilization frequencies between NPs and among doses were evaluated by the X2 test, and differences between NP diameters by ManneWhitney test or Kruskal Wallis test, due to their non-normal distribution (Kolmogorov Smirnov test). All statistics were performed using SPSS Statistics 19.0 for Windows.

Validity criteria fulfilled:

no

Conclusions:

The authors conclude that toxicity seems to be dpendent on size. However, they compare smaller ZnO aluminium doped with larger ZnO standard nano. Further, there is no effect from the zinc ion to compare to as only one concentration of zinc sulphate is tested. This study cannot be used for the assessment.

Reference 3

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

not specified

Principles of method if other than guideline:

Immobilization test according to OECD 202
Feeding inhibition test: Neonates (<24 h old) were separated from the main culture to other aquarium until they were 4 d old to 5 d old.
For each concentration tested, plus a control, 5 replicates with 5 individuals per treatment were used. Placed in 170-mL glass beakers with 100mL of the test substance and fed for 24 h with the algae P. subcapitata at a concentration of 50000 cell/ mL.

GLP compliance:

not specified

Remarks:

GLP compliance not specified in the publication

Specific details on test material used for the study:

Zinc oxide Nanosun NPs (Zn10 with approximately 30 nm) was supplied by Nanotrade as powder.
Zinc chloride (CAS number 7647-85-7; 98% purity) with a molar mass of 136.40 g/mol was purchased from Riedel-de Haën.

Analytical monitoring:

no

Vehicle:

no

Test organisms (species):

Daphnia magna

Details on test organisms:

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.
Daphnids were fed with Pseudokirchneriella subcapitata at a concentration of 300000 cell/mL and with 6mL/L of seaweed extract (equivalent to a dissolved organic carbon of 5mg/L).
Only the neonates from the third to fifth brood were used in toxicity tests to minimize variability.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Remarks on exposure duration:

24h for feeding inhibition test

Post exposure observation period:

4h for feeding inhibition test

Test temperature:

20+/-1°C

pH:

7.9+/-0.3

Nominal and measured concentrations:

Immobolization test
The nominal concentrations for ZnO-NPs ranged from 0.25 mg Zn/L to 10 mg Zn/L.
For zinc (in the form of ZnCl2), the nominal concentrations ranged from 0.2 mg Zn/L to 6.4 mg Zn/L.
Feeding inhibition test
Nominal test concentrations for ZnO 30 nm ranged from 1.2 mg Zn/L to 2.4 mg Zn/L.
For the zinc (in the form of ZnCl2), the nominal test concentrations ranged from 0.4 mg Zn/L to 12.8 mg Zn/L.

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

0.76 mg/L

Nominal / measured:

estimated

Conc. based on:

element

Remarks:

ZnCl2

Basis for effect:

mortality

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

1.02 mg/L

Nominal / measured:

estimated

Conc. based on:

element

Remarks:

nano ZnO

Basis for effect:

mortality

Duration:

4 h

Dose descriptor:

EC50

Effect conc.:

1.92 mg/L

Nominal / measured:

estimated

Conc. based on:

element

Remarks:

ZnCl2

Basis for effect:

behaviour

Remarks:

feeding

Duration:

4 h

Dose descriptor:

EC50

Effect conc.:

1.27 mg/L

Nominal / measured:

estimated

Conc. based on:

element

Remarks:

nano ZnO

Basis for effect:

behaviour

Remarks:

feeding

Reported statistics and error estimates:

All results were expressed as mg Zn/L 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 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.

Validity criteria fulfilled:

yes

Conclusions:

LC50 of both ZnO nano and ionic form are similar (OECD 202 test)
Feeding activities, measured as feeding rates (cell/mL), generally showed a decrease during the exposure and postexposure period with increasing concentrations for all the Zn forms studied. No mortality was observed during
the 24-h exposure during all tests. The smaller-sized ZnO-NPs (30 nm) appeared to be slightly more toxic.

Reference 4

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

no guideline followed

Principles of method if other than guideline:

There were three replicates for each treatment, each consisting of a 24-well multidish containing 8 T. japonicus nauplii placed individually in each well with 105 cells mL−1 of S. costatum provided as food. The duration of each test was 96 h, with test solutions being renewed once midway. Mortality was monitored daily and dead animals were removed

GLP compliance:

not specified

Remarks:

GLP compliance not specified in publication

Analytical monitoring:

yes

Vehicle:

no

Test organisms (species):

other aquatic crustacea: Tigriopus japonicus

Details on test organisms:

The copepod T. japonicus was sampled from a single high-shore rock pool at the Cape d’Aguilar Marine Reserve, Hong Kong (22°13′N, 114°12′E) in December 2008. The animals were acclimated in laboratory conditions for at least 2 days prior to the start of experiments, during
which the T. japonicus culture was fed with a diet mixture of S. costatum and the green alga Ulva lactua. For T. japonicus, gravid females
were selected from the culture and isolated in 24-well multidishes where they were fed with S. costatum. Nauplii
born within 24 h were then used for the tests.

Test type:

static

Water media type:

saltwater

Limit test:

no

Total exposure duration:

96 h

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

0.85 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Remarks:

nZnO

Basis for effect:

mortality

Remarks on result:

other: 0.78-0.94 (95% CI)

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

0.43 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Remarks:

bulk ZnO

Basis for effect:

mortality

Remarks on result:

other: 0.4-0.47 (95% CI)

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

0.68 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

dissolved

Remarks:

Zn (from nZnO)

Basis for effect:

mortality

Remarks on result:

other: 0.62-0.75 (95% CI)

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

1.14 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

dissolved

Remarks:

Zn from ZnSO4.7H2O

Basis for effect:

mortality

Remarks on result:

other: 0.86-1.5 (95% CI)

TEM images showed that the APS of nZnO was 26.2± 5.1 nm (mean ± s.d., n=102), which corroborated closely to the manufacturer’s information, while the APS of ZnO was significantly larger (student’s t test, t=25.907, p< 0.001 at 216.2±73.2 nm.

nZnO particles were more uniform in shape, being spherical to slightly elliptical, while ZnO particles were more angular and irregularly shaped. The hydrodynamic diameters (DH) of the metal oxides, on the other hand, portrayed a radically dissimilar picture. nZnO tended to form aggregates in seawater in the micrometer range (2.3±1.6 μm), which were even bigger than those formed by its bulk equivalent (1.7± 1.2 μm; t=4.183, p <0.001.

For both nZnO and ZnO, the equilibrium was achieved within 72 h after initial dispersion of both metal oxides in seawater. The dissolved Zn concentration for nZnO and ZnO was 3.7±0.6 and 1.6± 0.5 mg Zn L−1, respectively, at equilibrium. To our knowledge, this was the first study to investigate the solubility of nZnO in a saltwater environment. The solubility of ZnO at pH 8.0 was close to values (1.1– 2.5 mg Zn L−1) postulated in the literature. In contrast to previous research where the solubilities of nZnO and ZnO were found to be the same in distilled water, the current study showed that nZnO displays a higher solubility than its bulk equivalent in seawater, possibly because of its smaller size, larger surface area and curvature.

Validity criteria fulfilled:

yes

Conclusions:

Tests not done according to standard protocol but sufficient quality and considered useful for assessing acute aquatic ecotoxicity

Executive summary:

nZnO did not display the same toxicity as ZnO towards marine organisms. Plausible explanations included the change in DH as they formed aggregations, and the alteration to surface charge as pH of the environment increased from neutral to 8.0. Surface charge in turn affected the particle’s attachment onto organisms’ cells, and thus modified its toxicity. The toxicity of nZnO was shown to be influenced significantly (but not solely) by the release of Zn2+ ions into the solution, which is also supported by other research studies. In freshwater, the amount of dissolved Zn for the same concentration of nZnO and ZnO was similar, whereas in seawater, their solubility were altered by the change in pH, and this might have additionally contributed towards their differential toxicities.

Reference 5

Endpoint:

short-term toxicity to aquatic invertebrates

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:

There were three replicates for each treatment, each consisting of a 250-mL glass bowl containing ten E. rapax individuals. Two strips of nylon mesh (30×10 mm) were added to each glass bowl to provide a substrate for the amphipods. The duration of each test was 96 h, with test solutions being renewed once midway. Mortality was monitored daily and dead animals were removed.

GLP compliance:

not specified

Remarks:

GLP compliance not specified in publication

Analytical monitoring:

yes

Vehicle:

no

Test organisms (species):

other: Elasmopus rapax

Details on test organisms:

the amphipod E. rapax was obtained from the outdoor aquarium tanks at the Swire Institute of Marine Science (SWIMS), which were also supplied with running natural seawater from Cape d’Aguilar. The selected amphipods were measured linearly for the first to fourth thoracic segments because of their body curvature and averaged 0.39±0.05 mm in length (mean ± s.d.; n= 20). The animals were acclimated in laboratory conditions
for at least 2 days prior to the start of experiments.

Test type:

static

Water media type:

saltwater

Limit test:

no

Total exposure duration:

96 h

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

0.95 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

dissolved

Remarks:

Zn (from nano ZnO)

Basis for effect:

mortality

Remarks on result:

other: 0.55-1.66 (95% CI)

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

0.8 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

dissolved

Remarks:

Zn (from ZnSO4.7H2O)

Basis for effect:

mortality

Remarks on result:

other: 0.59-1.5 (95% CI)

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

1.19 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Remarks:

nano ZnO

Basis for effect:

mortality

Remarks on result:

other: 0.68-2.07 (95% CI)

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

0.37 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Remarks:

bulk ZnO

Basis for effect:

mortality

Remarks on result:

other: 0.25-0.53 (95% CI)

TEM images showed that the APS of nZnO was 26.2± 5.1 nm (mean ± s.d., n=102), which corroborated closely to the manufacturer’s information, while the APS of ZnO was significantly larger (student’s t test, t=25.907, p< 0.001 at 216.2±73.2 nm.

nZnO particles were more uniform in shape, being spherical to slightly elliptical, while ZnO particles were more angular and irregularly shaped. The hydrodynamic diameters (DH) of the metal oxides, on the other hand, portrayed a radically dissimilar picture. nZnO tended to form aggregates in seawater in the micrometer range (2.3±1.6 μm), which were even bigger than those formed by its bulk equivalent (1.7± 1.2 μm; t=4.183, p <0.001.

For both nZnO and ZnO, the equilibrium was achieved within 72 h after initial dispersion of both metal oxides in seawater. The dissolved Zn concentration for nZnO and ZnO was 3.7±0.6 and 1.6± 0.5 mg Zn L−1, respectively, at equilibrium. To our knowledge, this was the first study to investigate the solubility of nZnO in a saltwater environment. The solubility of ZnO at pH 8.0 was close to values (1.1– 2.5 mg Zn L−1) postulated in the literature. In contrast to previous research where the solubilities of nZnO and ZnO were found to be the same in distilled water, the current study showed that nZnO displays a higher solubility than its bulk equivalent in seawater, possibly because of its smaller size, larger surface area and curvature.

Validity criteria fulfilled:

yes

Conclusions:

Study not done according to guideline, considered supportive

Executive summary:

Toxicity mainly due to release of zinc ions

Reference 6

Endpoint:

short-term toxicity to aquatic invertebrates

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:

Sea urchin (Lytechinus pictus) embryos were exposed to a range of concentrations of nanomaterials and other Zn-containing compounds diluted into 0.45 m-filtered seawater (FSW) containing a final concentration of 0.2–0.3 mg/L alginate (Sigma, #A2158).

GLP compliance:

not specified

Remarks:

GLP compliance not specified in publication

Specific details on test material used for the study:

ZnO and Fe-doped ZnO NMs were synthesized by flame spray pyrolysis

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

Stock solutions (1 g/L) of metal oxide nanomaterials and the other Zn-containing compounds were prepared in de-ionized water and then sonicated in a Branson model 2510 sonic bath (Danbury, CT) for 30 min (max. 100 W). Each stock solution (unfiltered) was then diluted into 0.45 m filtered (to remove particulates) seawater containing 10 mg/L alginic acid as a source of dissolved organic carbon.

Test organisms (species):

other: Lytechinus pictus

Details on test organisms:

Embryos were exposed from the 2-cell stage until the pluteus larval stage (approximately 96 h after fertilization) in 12-well polystyrene culture plates, and incubated at 15 ◦C. When controls (0.2 mg/L alginate in 0.45 m filtered seawater) reached the pluteus stage, all samples were fixed with 0.1%
paraformaldehyde in seawater (final concentration) and assessed for normal development. The research by Kobayashi and Okamura was used as a reference for determining the normal pluteus larval morphology in control and Zn-exposed embryos

Test type:

static

Water media type:

saltwater

Limit test:

no

Total exposure duration:

96 h

Test temperature:

15°C

pH:

8.0 - 8.2

Nominal and measured concentrations:

Three initial concentrations
were evaluated: 0.1, 1.0 and 10 mg/L.

Details on test conditions:

Experiments were conducted at least three times, and for each experiment, treatments were always run in triplicate. For each replicate, 100 embryos were assessed for normal development using a Nikon AZ100 macrozoom stereo microscope at 40× magnification

Sea urchin (Lytechinus pictus) embryos were exposed to a range of concentrations of nanomaterials and other Zn-containing compounds diluted into 0.45 m-filtered seawater (FSW) containing a final concentration of 0.2–0.3 mg/L alginate (Sigma, #A2158). The pH of the FSW was 8.2, and was unaffected by 0.2–0.3 mg/L alginate.

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

ca. 98 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

dissolved

Remarks:

Zinc

Basis for effect:

morphology

Remarks on result:

other: ZnO nano

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

ca. 103 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

dissolved

Remarks:

Zinc

Basis for effect:

morphology

Remarks on result:

other: ZnO nano Fe doped

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

ca. 81 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

dissolved

Remarks:

Zinc

Basis for effect:

morphology

Remarks on result:

other: ZnO bulk

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

ca. 72 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

dissolved

Remarks:

Zinc

Basis for effect:

morphology

Remarks on result:

other: ZnSO4

Validity criteria fulfilled:

yes

Conclusions:

Tests done on standardised toxkit.Good quality and considered useful for assessing acute aquatic ecotoxicity.

Executive summary:

The toxicity of ZnO NM to developing sea urchin embryos appears to be largely attributable to the dissolution of Zn2+ into the seawater. At the concentrations used in our bioassay, ZnO is rapidly and completely solubilized. These results suggest that

the sea urchin embryos/larvae were exposed primarily to soluble Zn2+ throughout the 96 h developmental assay. Additionally,

the EC50 values for ZnO NM and bulk ZnO were not significantly different than the EC50 for ZnSO4·7H2O, a zinc salt that

is completely solubilized in less than 30 min. The morphological abnormalities that were observed were varied, but were consistent

with previous research that showed that Zn2+, in the g/L range, could cause a suite of abnormalities in developing sea urchin embryos

Reference 7

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study according to guideline but lack of documentation on the nano characteristics and no comparison to soluble salt, no information on agglomeration rate.

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Principles of method if other than guideline:

The suspensions were shaken at room temperature to obtain a final concentration of 1000 mg/L

GLP compliance:

not specified

Remarks:

GLP compliance not specified

Specific details on test material used for the study:

Nanoscale ZnO (nZnO) was purchased from Nanjing High Technology NANO Co., LTD (Nanjing, China); the bulk counterpart, ZnO/bulk, was purchased from The Third Chemical Regent Factory of Tianjin (Tianjin, China).

Analytical monitoring:

no

Vehicle:

no

Test organisms (species):

Daphnia magna

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Reference substance (positive control):

no

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.622 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

nZnO

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

1.511 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

nZnO

Basis for effect:

mortality

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.481 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

bulk ZnO

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

1.25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

bulk ZnO

Basis for effect:

mortality

Reported statistics and error estimates:

The 48-h EC50 and LC50, and their associated 95% confidence intervals (95% CI), were calculated using the EPA computer probit analysis program (Version 1.5). Statistical analyses were carried out using standard ANOVA techniques followed by Tukey’s honestly significant difference test (SPSS Ver. 14.0).
Differences were statistically significant when p<0.05

Validity criteria fulfilled:

yes

Conclusions:

Tests done according to standard protocol. Good quality and considered useful for setting the reference value for acute aquatic ecotoxicity

Executive summary:

nZnO was not found to be more toxic than its larger counterparts even though the published sizes of the different Zn particles ranged over 1 to 2 orders of magnitude. There is an assumption that particles of a suitable size (< 50,000 nm) are ingested by D. magna without any selective mechanism. Therefore, the sizes of the ZnO particles used in this study might not be sufficiently different to

significantly affect bioavailability or toxicity.

no comparison reported between the nano-form and the ion-form.

Reference 8

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

according to guideline

Guideline:

other: Standard Operational Procedures of Daphtoxkit FTM magna (1996)

Principles of method if other than guideline:

Crustacea ecotox assay was performed according to the Standard Operational Procedures of Daphtoxkit F™ magna (1996). Besides solutions with nanoparticles of ZnO also nanoparticles of TiO2 and CuO are tested in comparison as well as bulk oxides of the three metals and ZnSO4•7H2O. Additional tests with metal-specific biosensors are conducted in order to differentiate between toxic effects of metal oxides per se and solubilised metal ions.

GLP compliance:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Stock suspensions in Milli-Q (40g/L) were sonicated for 30 min.
- stock solution of ZnSO4.7H2O not sonicated but vortexed before use

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: water flea (Crustacea), neonates

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test temperature:

20°C

pH:

7.3 - 7.8

Details on test conditions:

TEST SYSTEM
- No. of vessels per concentration (replicates): 4 replicates were done.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Test compound was diluted in synthetic freshwater (diluent included in the test kit, also used as a control).

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

7.1 mg/L

Nominal / measured:

nominal

Conc. based on:

element

Remarks:

Zinc from bulk ZnO

Basis for effect:

mortality

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

2.6 mg/L

Nominal / measured:

nominal

Conc. based on:

element

Remarks:

Zinc from nano ZnO

Basis for effect:

mortality

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

1.4 mg/L

Nominal / measured:

nominal

Conc. based on:

element

Remarks:

Zinc from ZnSO4.7H2O

Basis for effect:

mortality

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

1.2 mg/L

Nominal / measured:

nominal

Conc. based on:

element

Remarks:

Zinc from bulk ZnO

Basis for effect:

mortality

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.4 mg/L

Nominal / measured:

nominal

Conc. based on:

element

Remarks:

Zinc from nano ZnO

Basis for effect:

mortality

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.45 mg/L

Nominal / measured:

nominal

Conc. based on:

element

Remarks:

Zinc from ZnSO4.7H2O

Basis for effect:

mortality

All Zn formulations were toxic: L(E)C50 (mg/L) for bulk ZnO, nanoZnO and ZnSO4 •7H2O: 8.8, 3.2, 6.1. The toxicity was due to solubilized Zn ions as proved with recombinant Zn-sensor bacteria.

Validity criteria fulfilled:

yes

Conclusions:

Tests done on standardised toxkit.Good quality and considered useful for assessing acute aquatic ecotoxicity.

Executive summary:

Toxicity of all Zn compounds were quite comparable. As compared with toxicity of ZnSO4, not all the Zn bioavailable for E.coli Zn-sensors from nano and bulk ZnO proved bioavailable for daphnids.

Comparison of ecotox levels between the nano form and the ionic Zn2+ form is not possible because only nominal concentrations are reported.

Reference 9

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: Conflictual results

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

yes

Remarks:

additional endpoints

Principles of method if other than guideline:

Assessment of bioaccumulation of ZnO nanoparticles

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:Suspensions of ZnO NPs and bulk ZnO were prepared using standard operation protocols developed by the National Physics Laboratory (http://www.nanotechia-prospect.org). The NPs and bulk powders were made into a paste by adding one or two drops of deionized water (DIW) to 25 mg of particle mass and the mixture stirred with a metal spatula. Using a glass Pasteur pipet, 9−10 drops of DIW were then added slowly, continuing to mix the paste. The remaining DIW (250 mL, except for the few drops used to make the paste) was added to the paste and the suspension sonicated twice for 10 s using an ultrasonic probe (Cole Parmer 130 W Ultrasonic Processor). Bulk and ZnO NM suspensions were made freshly, immediately before dosing the exposure vessels.

- Controls: D.magna media


- Evidence of undissolved material (e.g. precipitate, surface film, etc):

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: D.Magna
- Strain:
- Source:University of Birmingham


- Feeding during test No
- Food type:
- Amount:
- Frequency:

ACCLIMATION
- Acclimation period:
- Acclimation conditions (same as test or not): same as test
- Type and amount of food: Cholorella vulgaris and Baker`s yeast
- Feeding frequency: daily
- Health during acclimation (any mortality observed):

QUARANTINE (wild caught)
- Duration:
- Health/mortality:

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Remarks on exposure duration:

and 24 h

Post exposure observation period:

24 h after exposure feeding rate assessed

Test temperature:

20°C

pH:

7.5

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 ml beakers
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: 100 ml of test media
- Aeration: Yes
- Type of flow-through (e.g. peristaltic or proportional diluter): N/A
- Renewal rate of test solution (frequency/flow rate):N/A
- No. of organisms per vessel: 30
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates):3
- No. of vessels per vehicle control (replicates): no vehicle used
- Biomass loading rate:

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
- Total organic carbon:
- Particulate matter:
- Metals:
- Pesticides:
- Chlorine:
- Alkalinity:
- Ca/mg ratio:
- Conductivity:
- Culture medium different from test medium:
- Intervals of water quality measurement:

OTHER TEST CONDITIONS
- Adjustment of pH:
- Photoperiod: 16 h light to 8 h dark
- Light intensity:

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :

TEST CONCENTRATIONS
- Spacing factor for test concentrations:
- Justification for using less concentrations than requested by guideline:
- Range finding study
- Test concentrations:
- Results used to determine the conditions for the definitive study:

Reference substance (positive control):

yes

Remarks:

Micro-size Zno and ionic ZnO used as reference material

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

1.55 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

nano ZnO

Basis for effect:

mortality

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

3.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

bulk ZnO

Basis for effect:

mortality

Results with reference substance (positive control):

- Results with reference substance valid? Ionic Zn
- Mortality: yes 48 h acute testing
- LC50: 3.3 mg/L
- Other:

Reported statistics and error estimates:

Anova

LC values for an acute (48h) exposure of ZnO MNP and bulk ZnO to 3‐d old D. magna (EPA Probit analysis).

ZnCl2		95%confidence interval.		ZnO MNP		95%  confidence interval.
	Exposureconc(mgl‐ 1)	lower	Upper		Exposureconc(mgl‐1	lower	Upper
LC1	0.887	0.62	1.143	LC1	0.282	0.183	0.386
LC5	1.305	0.989	1.596	LC5	0.465	0.333	0.595
LC10	1.604	1.265	1.91	LC10	0.608	0.457	0.752
LC15	1.844	1.493	2.159	LC15	0.728	0.565	0.882
LC50	3.321	2.932	3.713	LC50	1.557	1.335	1.796
LC85	5.98	5.294	6.944	LC85	3.33	2.827	4.086
LC90	6.873	6.015	8.149	LC90	3.987	3.33	5.031
LC95	8.447	7.238	10.377	LC95	5.205	4.224	6.88
LC99	12.436	10.157	16.457	LC99	8.582	6.543	12.485

The feeding rate (uptake of the algae Chlorella vulgaris) was measured as a sublethal effect of exposure of D. Magna to ZnO MNP (Micronisers, APS 30 nm). Organisms exposed to ZnO MNP had a lower feeding rate (reduction of up to 30% at 1 mg l‐1 ) compared to bulk ZnO and soluble zinc . Scanning electron micrographs suggested that the decrease in feeding rate was not related to a physical impairment or obstruction as a result of binding of ZnO MNP or ZnO MNP aggregates to the antenna nor thoracic appendages of the organisms.

See attached document section for graphs and images

Validity criteria fulfilled:

yes

Executive summary:

Acute exposure (48h) of D. magna to ZnO MNPs (modified OECD test guideline 202) caused a concentration‐dependent increase on mortality (LC50 1.55 mg l‐1).

D. magna exposed to sublethal concentrations of ZnO MNPs had a lower feeding rate compared to bulk ZnO and soluble zinc. This did not appear to be related to physical impairment or obstruction of the organism’s feeding apparatus.

Bioimaging using Coherent Anti‐stokes Raman Scattering (CARS) and optical light microscopy confirmed that D. magna ingested large quantities of both MNPs, but in the case of CeO2 MNPs the high uptake did not affect survival.

Reference 10

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable, well documented publication/study report which meets basic scientific principles

Qualifier:

according to guideline

Guideline:

other: Standard Operational Procedures of Thamnotoxkit FTM (1995)

Principles of method if other than guideline:

Crustacea ecotox assay was performed according to the Standard Operational Procedures of Thamnotoxkit F™ (1995). Besides solutions with nanoparticles of ZnO also nanoparticles of TiO2 and CuO are tested in comparison as well as bulk oxides of the three metals and ZnSO4•7H2O. Additional tests with metal-specific biosensors are conducted in order to differentiate between toxic effects of metal oxides per se and solubilised metal ions.

GLP compliance:

no

Analytical monitoring:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Stock suspensions in Milli-Q (40g/L) were sonicated for 30 min.
- ZnSO4.7H2O solution not sonicated but vortexed before use

Test organisms (species):

other aquatic crustacea: Thamnocephalus platyurus

Details on test organisms:

TEST ORGANISM
- Common name: fairy shrimp (Crustacea)

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

24 h

Test temperature:

25°C

pH:

7.3 - 7.8

Details on test conditions:

TEST SYSTEM
- No. of vessels per concentration (replicates): 3 replicates were done.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Test compound was diluted in synthetic freshwater (diluent included in the test kit, also used as a control).

Duration:

24 h

Dose descriptor:

LC50

Effect conc.:

0.19 mg/L

Nominal / measured:

nominal

Conc. based on:

element

Remarks:

Zinc from bulk ZnO

Basis for effect:

mortality

Duration:

24 h

Dose descriptor:

LC50

Effect conc.:

0.14 mg/L

Nominal / measured:

nominal

Conc. based on:

element

Remarks:

Zinc from nano ZnO

Basis for effect:

mortality

Duration:

24 h

Dose descriptor:

LC50

Effect conc.:

0.22 mg/L

Nominal / measured:

nominal

Conc. based on:

element

Remarks:

Zinc from ZnSO4.7H2O

Basis for effect:

mortality

Duration:

24 h

Dose descriptor:

NOEC

Effect conc.:

0.04 mg/L

Nominal / measured:

nominal

Conc. based on:

element

Remarks:

Zinc from bulk ZnO

Basis for effect:

mortality

Duration:

24 h

Dose descriptor:

NOEC

Effect conc.:

0.02 mg/L

Nominal / measured:

nominal

Conc. based on:

element

Remarks:

Zinc from nano ZnO

Basis for effect:

mortality

Duration:

24 h

Dose descriptor:

NOEC

Effect conc.:

0.25 mg/L

Nominal / measured:

nominal

Conc. based on:

element

Remarks:

Zinc from ZnSO4.7H2O

Basis for effect:

mortality

All Zn formulations were toxic: L(E)C50 (mg/L) for bulk ZnO, nanoZnO and ZnSO4•7H2O: 0.24, 0.18, 0.98. The toxicity was due to solubilized Zn ions as proved with recombinant Zn-sensor bacteria.

Validity criteria fulfilled:

yes

Conclusions:

Tests done on standardised toxkit.Good quality and considered useful for assessing acute aquatic ecotoxicity.

Executive summary:

As a rule, toxicity of all Zn compounds were quite comparable. Effects of ZnO in T. platyurus were due to Zn ions, as toxicity of all tested zinc compounds was similar when expressed as mg Zn /l.

Comparison of ecotox levels between the nano form and the ionic Zn2+ form is not possible because nominal concnetrations are reported only.

Reference 11

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: No comparison woth bulk or soluble zinc salt

Qualifier:

according to guideline

Guideline:

other: APHA (2005)

GLP compliance:

not specified

Specific details on test material used for the study:

Zinc oxide nanoparticles (ZnONPs) dry powder (Product No. 677450, APS: 50 nm and SSA: >10.8 m2/g) was purchased from M/s. Sigma–Aldrich, St. Louis, MO, USA.

Analytical monitoring:

yes

Vehicle:

no

Test organisms (species):

other: Lymnaea luteolo L

Details on test organisms:

Individuals of adult snail (L. luteola) of similar size and weight were carefully collected from non-contaminated artificial fish culture ponds situated at Indian Institute of Toxicology Research (IITR), Gheru Campus, Lucknow, and transferred to the laboratory. They were maintained in glass aquaria. Snails were acclimatized to laboratory conditions for 2 weeks before experimentation, at temperature 21±1 ◦C and fed daily ad libitum with thoroughly washed freshwater green aquatic plant (Marsilia sp.) leaves. They had an average wet weight of 500 mg (range 350–650 mg) and shell length
22 mm (range 19–25 mm).

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

160-180 mg/L

Test temperature:

25.8-27.4°C

pH:

7.05-8.1

Dissolved oxygen:

6-8.2 mg/L

Salinity:

46-56 mg Cl/L

Nominal and measured concentrations:

0-5-10-20-40-60-80-100 µg/ml

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

42.67 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

ZnO NP

Basis for effect:

mortality

Reported statistics and error estimates:

ANOVA, p<0.01

The UV–vis spectrophotometer showed a sharp absorption band at 390 nm. The crystal structure of ZnONPs was characterized by X-ray diffraction (PANalytical X’Pert Pro X-ray diffractometer) with Cu K radiation ( = 0.15418 nm). X-ray diffraction patterns of ZnONPs indicates two peaks at 2 = 31.67◦, 34.31◦, 36.14◦, 47.40◦, 56.52◦, 62.73◦, 66.28◦, 67.91◦, 69.03◦, 72.48◦ and 78.64◦ were assigned to (1 0 0), (0 0 2), (1 0 1), (1 0 2), (1 1 0), (1 0 3), (2 0 0), (1 1 2), (2 0 1), (0 0 4) and (2 0 2) of ZnONPs, indicating that the samples were polycrystalline wurtzite structure (Zincite, JCPDS 5-0664). No characteristic peaks of any impurities were detected, suggesting that high-quality ZnONPs were synthesized. The average crystallite size (d) of ZnONPs was estimated by Scherrer’s formula (Patterson, 1939). d = K ˇ cos where K = 0.9 is the shape factor, is the X-ray wavelength of Cu K radiation (1.54A˚ ), is the Bragg diffraction angle and ˇ is the FWHM of the respective diffraction peak. The average crystallite size of ZnONPs was found to be around 22 nm. Fig. 1C shows the typical TEM image of ZnONPs. This picture exhibits that the majority of the particles were in polygonal shape with smooth surfaces. TEM average diameter was calculated from measuring over 100 particles in random fields of TEM view. The average TEM diameter of ZnONPs was around 22 nm supporting the XRD data. Fig. 1D represents the frequency of size (nm) distribution of ZnONPs. The average hydrodynamic size and zeta potential of ZnONPs in water determined by DLS were 264.8 nm and −15.3 mV, respectively.

The released Zn2+ concentrations in test solution of ZnONPs were measured. Zn2+ concentrations in test water were found to be 1.098 g/ml at lowest and 2.040 g/ml at highest exposure solution

Validity criteria fulfilled:

yes

Conclusions:

Tests done according to standard protocol. Good quality and considered useful for assessing acute aquatic ecotoxicity

Reference 12

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

no guideline available

Principles of method if other than guideline:

test according to standardised "Thamnotoxkit F"

GLP compliance:

not specified

Remarks:

GLP compliance not specified in publication

Specific details on test material used for the study:

70nm

Analytical monitoring:

yes

Vehicle:

no

Details on test solutions:

tests were done in artificial water and 6 natural river waters.

Test organisms (species):

other aquatic crustacea: Thamnocephalus platyurus

Details on test organisms:

Thamnotoxkit F

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

24 h

Hardness:

Ca++ in natural waters varied between 124 and 58 mg/l; artificial medium CaCl2.2H20: 294mg/l

Test temperature:

25°C

pH:

pH of natural waters varied between 7.5 and 8.2, artificial water: 7.8

Nominal and measured concentrations:

Zn background of natural waters baried between 1.4 and 3.1 mg (total)/l

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

0.14 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

dissolved

Remarks:

Zinc from nano ZnO

Basis for effect:

mortality

Remarks on result:

other: artificial water

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

0.19 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

dissolved

Remarks:

Zinc from bulk ZnO

Basis for effect:

mortality

Remarks on result:

other: artificial water

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

0.22 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

dissolved

Remarks:

Zinc from ZnSO4.7H2O

Basis for effect:

mortality

Remarks on result:

other: artificial water

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

>= 1.1 - <= 6 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

dissolved

Remarks:

Zinc from nano ZnO

Basis for effect:

mortality

Remarks on result:

other: natural waters

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

ca. 0.5 - ca. 4 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

dissolved

Remarks:

Zinc from bulk ZnO

Basis for effect:

mortality

Remarks on result:

other: natural waters

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

>= 0.61 - <= 1.7 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

dissolved

Remarks:

Zinc from ZnSO4.7H2O

Basis for effect:

mortality

Remarks on result:

other: natural waters

Details on results:

Crustacean Thamnocephalus platyurus, 24 h LC50 (mean±SD, mg metal/l)

Nano ZnO bulk ZnO ZnSO4.7H2O
AFWa 0.14±0.02 0.19±0.03 0.22±0.06
River 1 1.1±0.23 >4 0.92±0.11
River 2 6±0.71 1.9±0.46 1.6±0.2
River 3 3.6±0.74 3±0.37 0.61±0.26
River 4 1.5±0.32 <0.5 0.75±0.06
River 5 5.3±0.5 2.1±0.19 1.1±0.4
River 6 1.4±0.35 1.2±0.16 1.7±0.88

Reported statistics and error estimates:

One-way analysis of variance (ANOVA) followed by t-testswas used to determine statistical significance of the differences between toxic effects of the compounds in different test media. The differences were considered significant, when p < 0.05.

Validity criteria fulfilled:

yes

Conclusions:

Tests done on standardised toxkit. Good quality and considered useful for assessing acute aquatic ecotoxicity.

Executive summary:

Standardised toxkit test on Thamnocephalys platyurus to check the effect of ZnO nanoparticles. As in standard test conditions, the toxic effect of ZnO NPs in natural waters was mainly due to dissolved metal ions. Thus, to understand the mechanisms of ecotoxicological action of metal oxides NPs and its ecological consequences, solubility and speciation are the crucial aspects.

Description of key information

The present short-term acute aquatic toxicity database for nano-ZnO, covers 5 invertebrates (2 freshwater and 3 marine) species. The full set of EC₅₀ values are presented in the attached tables.

Additional information

Nano ZnO data set

The present short-term acute aquatic toxicity database for nano-ZnO, covers 5 invertebrates (2 freshwater and 3 marine) species. For reasons of comparison, in the attached tables, the data are presented as normalized values with the ecotoxicity value observed for the soluble zinc salts (Zn²⁺) as reference (EC_x = 100%). The ratio EC_x Zn²⁺/EC_x nano ZnO is provided. The detail of the studies is in the relevant IUCLID sections. The full set of EC₅₀ values are presented in the attached tables.