Zinc oxide

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Version / remarks:

2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Lot/Batch number: 1961
Purity: 99.8 %
Zn in ZnO: 80.3%
Surface area (BET): 51 m²/g
State of matter and appearance: white powder, nanoparticle

Analytical monitoring:

yes

Details on sampling:

i) ‘Total’ zinc - an unfiltered sample of the test medium,
ii) “First filtered” zinc - a 0.2 µm filtered sample of the test medium. Although assumed to potentially contain nano and dissolved, it is hereafter termed ‘dispersed’ zinc for ease of reference and,
iii) “Second filtered” zinc - sample from the test medium which has been further separated by a 3 kDa centrifugal filtration – assumed to be dissolved, so hereafter termed ‘dissolved’ zinc.

All samples were stabilised by acidification and if needed stored at 4 °C ± 3 °C until further analysis. The samples will be discarded after finalisation of the study.

Vehicle:

no

Details on test solutions:

The preparation of the test item solutions followed the procedures described in the OECD TG 318. An appropriate amount of the test item was weighed into a glass beaker with a nominal volume of 250 mL, pre-wetted with 1 mL dilution water and left in the form of wet paste for 24 h to insure the proper interaction of the nanomaterial surface with the water. After 24 h of pre-wetting the resulted wet paste was dispersed in 149 mL of dilution water, thus providing a stock dispersion with known nanomaterial concentration.

Test organisms (species):

Daphnia magna

Details on test organisms:

The test organisms were young Daphnia magna, 4 – 24 hours old. Origin of the cladocerans is the German Federal Environment Agency, Institut für Wasser-, Boden- und Lufthygiene. Specimens used in the test are bred in the laboratory of the Fraunhofer IME for over 15 years.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

300-320 mg CaCO3/L

Test temperature:

19.5 – 20.0 °C

pH:

8.15 - 9.40

Dissolved oxygen:

The dissolved oxygen saturation was between 8.3 mg/L and 11.6 mg/L

Salinity:

na

Conductivity:

696 µS/cm

Nominal and measured concentrations:

Total Zn (µg/l): Nominal: Control, 76.9, 131, 222, 378, 642
Total Zn (µg/l): measured (Time Weighted Mean): 10.4, 61.6, 105, 171, 248, 352

Dispersed Zn (µg/l): Nominal: Control, 76.9, 131, 222, 378, 642
Dispersed (µg/l): measured (Time Weighted Mean): 8.51, 22.8, 49.2, 93.1, 125, 151

Dissolved Zn (µg/l): Nominal: Control, 76.9, 131, 222, 378, 642
Dissolved Zn (µg/l): measured (Time Weighted Mean): 6.55, 24.1, 49.6, 87.2, 121, 139

Details on test conditions:

Refer to report.

Reference substance (positive control):

no

Key result

Duration:

21 d

Dose descriptor:

EC10

Remarks:

Total fraction (non-filtered)

Effect conc.:

127 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

element (total fraction)

Basis for effect:

reproduction

Remarks:

Offspring per introduced parent

Key result

Duration:

21 d

Dose descriptor:

EC10

Remarks:

Nominal

Effect conc.:

195 µg/L

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

reproduction

Remarks:

Offspring per introduced parent

Duration:

21 d

Dose descriptor:

EC10

Remarks:

Dispersed fraction (0.2 µm filter)

Effect conc.:

64.3 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

element (dissolved fraction)

Basis for effect:

reproduction

Remarks:

Offspring per introduced parent

Duration:

21 d

Dose descriptor:

EC10

Remarks:

Dissolved fraction (3 kDa filter)

Effect conc.:

60.8 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

element (dissolved fraction)

Basis for effect:

reproduction

Remarks:

Offspring per introduced parent

Reported statistics and error estimates:

For each endpoint, the NOEC, LOEC, and, if possible, the EC50 and EC10 is determined. A LOEC is calculated by using ANOVA followed by Student-t test, Fisher`s Exact Binomial test, Dunnett’s or Williams’ test or an appropriate non-parametric test. When the test results show a concentration-response relationship, the data is analysed by regression to determine the EC50 including the 95% confidence interval as well as the EC10 using Probit-analysis assuming log-normal distribution of the values.
The computer software ToxRat® will be used for statistical evaluations.
The evaluation of the concentration-effect-relationships and the calculations of effect concentrations were based on the time weighted mean (TWM) concentrations for each fraction (total, dispersed and dissolved), since the measured concentrations varied by more than 20% from the nominal concentrations throughout the test.

A filter test was performed to investigate the influence of the filtration process, specifically the amount of filtered solution through one filter, on the measured Zn concentration in the fraction below 0.2 µm (dispersed Zn). The use of one filter (0.2 µm PES) for a sample of 40 mL was compared to the use of four filters (0.2. µm PES) for a sample of 40 mL (as done in the definitive Daphnia magna reproduction test), which was devided in subsamples of 10 mL each, thereby reducing the amount of sample filtered though one individual filter. The procedure of the test is described in detail in chapter 4.3.

After filtration of one sample of 40 mL volume, in samples of nominal concentrations of 76.9 and 642 µg Zn/L, 9.49 and 117 µg Zn/L were measured, corresponding to 12.3 and 18.2 % of nominal. After rinsing the used filters with pure test medium, 5.77 and 28.9 µg Zn/L were measured indicating, that additional Zn was released from the filters.

Using a step-wise approach with filtration of max. 10 mL resulted in measured concentrations between 20.7 and 22.0 µg Zn/L (26.9 – 28.6 % of nominal) for 76.9 µg Zn/L and 195 – 204 µg Zn/L (30.3 – 31.7 % of nominal) for 642 µg Zn/L.

Overall, the results for the total fraction show that the test media preparation worked well with recovery rates between 109 – 116 % of nominal.

In addition, it was demonstrated, that using a step-wise approach with four different filters to process a volume of 40 mL lead to slightly higher concentrations of the dispersed Zn fraction (26.9 – 31.7 % of nominal) compared to the filtration through only one filter (12.3 – 18.2 % of nominal) which was applied during the definitive test

Validity criteria fulfilled:

yes

Remarks:

The test fulfils all validity criteria of the OECD guideline 211 as: Mortality in controls (0%) did not exceed 20%. The mean number of offspring in the control within the 21 days (112.3) was above the criterion of 60 / female.

Conclusions:

Based on the evaluation performed on TWM concentrations of the total zinc fraction, for reproduction per introduced parent daphnid an EC10 of 195.0 µg nominal Zn/L, an EC10 of 127.0 µg total Zn/L and a NOEC of 49.6 µg total Zn/L was determined .

Executive summary:

During the 21-day exposure period, the test item had significant effects on the investigated endpoints of reproduction, immobilisation, length and intrinsic rate r of population increase r of the test organisms Daphnia magna.
Since a significant effect on the immobility of the daphnids was observed, the reproduction per introduced parent daphnid is the relevant chronic endpoint considered in the present study.

It is worth noting that no nano was determined (dispersed - truly dissolved) in any of the concentrations of nano material added during the test; therefore, it is justified to use the 'total EC10' value of 127.0 total Zn µg/L.

In addition, the 'Filter Test', at a low exposure concentration (76.9 µg Zn/L) revealed that x2.26 more dispersed Zn passed through x4 filters, when compared to the same volume of media being passed through x1 filter (as per the test). When applying this ratio to the dispersed EC10 of 64.3 µg/L, a filter-corrected EC10 value of 145.1 µg/L was calculated.

Reference 2

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Version / remarks:

2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Lot/Batch number: EHRH1406
Purity: 98.2 %
Zn in ZnO: 80.3%
Average particle size: 0.1 µm
State of matter and appearance: white powder, nanoparticle

Analytical monitoring:

yes

Details on sampling:

i) ‘Total’ zinc - an unfiltered sample of the test medium,
ii) “First filtered” zinc - a 0.2 µm filtered sample of the test medium. Although assumed to potentially contain nano and dissolved, it is hereafter termed ‘dispersed’ zinc for ease of reference and,
iii) “Second filtered” zinc - sample from the test medium which has been further separated by a 3 kDa centrifugal filtration – assumed to be dissolved, so hereafter termed ‘dissolved’ zinc.

All samples were stabilised by acidification and if needed stored at 4 °C ± 3 °C until further analysis. The samples will be discarded after finalisation of the study.

Vehicle:

no

Details on test solutions:

The preparation of the test item solutions followed the procedures described in the OECD TG 318. An appropriate amount of the test item was weighed into a glass beaker with a nominal volume of 250 mL, pre-wetted with 1 mL dilution water and left in the form of wet paste for 24 h to insure the proper interaction of the nanomaterial surface with the water. After 24 h of pre-wetting the resulted wet paste was dispersed in 149 mL of dilution water, thus providing a stock dispersion with known nanomaterial concentration.

Test organisms (species):

Daphnia magna

Details on test organisms:

The test organisms were young Daphnia magna, 4 – 24 hours old. Origin of the cladocerans is the German Federal Environment Agency, Institut für Wasser-, Boden- und Lufthygiene. Specimens used in the test are bred in the laboratory of the Fraunhofer IME for over 15 years.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

310-340 mg CaCO3/L

Test temperature:

19.0 – 20.0 °C

pH:

7.94 – 9.53

Dissolved oxygen:

The dissolved oxygen saturation was between 7.9 mg/L and 12.3 mg/L

Salinity:

na

Conductivity:

696 µS/cm

Nominal and measured concentrations:

Total Zn (µg/l): Nominal: Control, 76.9, 131, 222, 378, 642
Total Zn (µg/l): measured (Time Weighted Mean): 6.12, 63.1, 114, 169, 254, 386

Dispersed Zn (µg/l): Nominal: Control, 76.9, 131, 222, 378, 642
Dispersed (µg/l): measured (Time Weighted Mean): 12.8, 20.1, 47.0, 75.2, 102, 134

Dissolved Zn (µg/l): Nominal: Control, 76.9, 131, 222, 378, 642
Dissolved Zn (µg/l): measured (Time Weighted Mean): 12.1, 17.4, 42.9, 68.5, 95.2, 131

Details on test conditions:

Refer to report.

Reference substance (positive control):

no

Key result

Duration:

21 d

Dose descriptor:

EC10

Remarks:

Total fraction (non-filtered)

Effect conc.:

93.4 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

element (total fraction)

Basis for effect:

reproduction

Remarks:

Offspring per introduced parent

Key result

Duration:

21 d

Dose descriptor:

EC10

Remarks:

Nominal

Effect conc.:

102 µg/L

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

reproduction

Remarks:

Offspring per introduced parent

Duration:

21 d

Dose descriptor:

EC10

Remarks:

Dispersed fraction (0.2 µm filter)

Effect conc.:

38.7 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

element (dissolved fraction)

Basis for effect:

reproduction

Remarks:

Offspring per introduced parent

Duration:

21 d

Dose descriptor:

EC10

Remarks:

Dissolved fraction (3 kDa filter)

Effect conc.:

35.1 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

element (dissolved fraction)

Basis for effect:

reproduction

Remarks:

Offspring per introduced parent

Reported statistics and error estimates:

For each endpoint, the NOEC, LOEC, and, if possible, the EC50 and EC10 is determined. A LOEC is calculated by using ANOVA followed by Student-t test, Fisher`s Exact Binomial test, Dunnett’s or Williams’ test or an appropriate non-parametric test. When the test results show a concentration-response relationship, the data is analysed by regression to determine the EC50 including the 95% confidence interval as well as the EC10 using Probit-analysis assuming log-normal distribution of the values.
The computer software ToxRat® will be used for statistical evaluations.
The evaluation of the concentration-effect-relationships and the calculations of effect concentrations were based on the time weighted mean (TWM) concentrations for each fraction (total, dispersed and dissolved), since the measured concentrations varied by more than 20% from the nominal concentrations throughout the test.

A filter test was performed to investigate the influence of the filtration process, specifically the amount of filtered solution through one filter, on the measured Zn concentration in the fraction below 0.2 µm (dispersed Zn). The use of one filter (0.2 µm PES) for a sample of 40 mL was compared to the use of four filters (0.2. µm PES) for a sample of 40 mL (as done in the definitive Daphnia magna reproduction test), which was devided in subsamples of 10 mL each, thereby reducing the amount of sample filtered though one individual filter. The procedure of the test is described in detail in chapter 4.3.

After filtration of one sample of 40 mL volume, in samples of nominal concentrations of 76.9 and 642 µg Zn/L, 8.79 and 127 µg Zn/L were measured, corresponding to 11.4 and 19.7 % of nominal. After rinsing the used filters with pure test medium, 9.29 and 43.0 µg Zn/L were measured indicating, that additional Zn was released from the filters.

Using a step-wise approach with filtration of max. 10 mL resulted in measured concentrations between 17.7 and 19.9 µg Zn/L (23.0 – 25.9 % of nominal) for 76.9 µg Zn/L and 179 – 185 µg Zn/L (27.8 – 28.8 % of nominal) for 642 µg Zn/L.

Overall, the results for the total fraction show that the test media preparation worked well with recovery rates between 111 – 113 % of nominal.

In addition, it was demonstrated, that using a step-wise approach with four different filters to process a volume of 40 mL lead to slightly higher concentrations of the dispersed Zn fraction (23.0 – 28.8 % of nominal) compared to the filtration through only one filter (11.4 – 19.7 % of nominal) which was applied during the definitive test.

Validity criteria fulfilled:

yes

Remarks:

The test fulfils all validity criteria of the OECD guideline 211 as: Mortality in controls (0%) did not exceed 20%. The mean number of offspring in the control within the 21 days (109.3) was above the criterion of 60 / female.

Conclusions:

Based on the evaluation performed on TWM concentrations of the total zinc fraction, for reproduction per introduced parent daphnid an EC10 of 102.0 µg nominal Zn/L, an EC10 of 93.4 µg total Zn/L and a NOEC of 63.1 µg total Zn/L was determined.

Executive summary:

During the 21-day exposure period, the test item had significant effects on the investigated endpoints of reproduction, immobilisation, length and intrinsic rate r of population increase r of the test organisms Daphnia magna.
Since a significant effect on the immobility of the daphnids was observed, the reproduction per introduced parent daphnid is the relevant chronic endpoint considered in the present study. 

It is worth noting that no nano was determined (dispersed - truly dissolved) in any of the concentrations of nano material added during the test; therefore, it is justified to use the 'total EC10' value of 93.4 µg/L.

In addition, the 'Filter Test', at a low exposure concentration (76.9 µg Zn/L) revealed that x2.14 more dispersed Zn passed through x4 filters, when compared to the same volume of media being passed through x1 filter (as per the test). When applying this ratio to the dispersed EC10 of 38.7 µg/L, a filter-corrected EC10 value of 82.8 µg/L was calculated.

Description of key information

The data newly generated on D. subspicatus and D. magna did not show evidence for additional toxicity of ZnO-nanoforms tested.

Additional information

Evaluation requests 4&5: ZnO-nanoform ecotoxicity testing on D . subspicatus and D. magna

As part of the testing programme requested by the Decision of July 9th, 2019 on Zinc Oxide (ECHA 2019), chronic aquatic toxicity testing has been performed on the freshwater algae Desmodesmus subspicatus, and the invertebrate Daphnia magna (requests 4 and 5 of the Decision). The ecotoxic effect of the selected nanoforms were compared to ZnCl2  in the same conditions to assess any possible additional effect than that of the ion.

It was evident from the suite of ecotoxicity testing on D. subspicatus and D. magna, that there were no nanoparticles dispersed in solution, instead they rapidly formed agglomerates larger than 0.2 µm (i.e., no difference observed in any of the dispersed vs. dissolved fractions).

Two main observations in the ecotoxicity tests lead to conclude to no additional effect of the nanoforms:
- ECx values of the nanoforms expressed as nominal or total zinc are not different from ZnCl2, or higher than the soluble reference compound ZnCl2. This is observed for both the alga and the daphnid.
- ECx results expressed as dispersed and soluble fractions are very similar throughout the tests. This observation supports the absence of nanoparticle shortly after entering the test solution, as shown also by the transformation/dissolution results (Cooper et al 2021). 

Considering the variability observed in these tests, the differences with the reference compound ZnCl2 within a factor 2 (corresponding to variation of 38% on the mean value) reflect inherent test variability, rather than effect. The reverse pattern of results observed in some cases (e.g. slightly lower EC10 versus higher EC50) is also interpreted as reflecting test variability, rather than resulting from a nanoparticle effect.

In the algae tests, the results on the hydrophobic coated nanoform 369 were unreliable. Precipitates were visible during the algae test and were examined in more detail. It appears that the organic triethoxy(octyl) silane coating of the 369 nanoform, dissolved and resulted in a severe disturbance of the distribution of zinc over different fractions in the test, including precipitated layers. This phenomenon made unreliable both the measurements of zinc in the test solution, and, as a consequence, the ECx values calculated on measured zinc.

In the D. magna tests, the 1st filtration step over the 0.2 µm filter resulted in a compelling loss of zinc. This test artefact had a significant effect on the calculated ECx values of the dispersed and dissolved fractions. Finally, the hydrophobic coated 369 nanoform hardly dissolved in the (static) D. magna test, resulting in no effect at 100 mg/L.

In conclusion, the data generated on D. subspicatus and D. magna did not show any evidence for additional toxicity of the ZnO-nanoforms tested.

More comprehensive summary data, discussion of the choice of testing materials, discussion of the results and conclusions is provided in the Industry document “Summary background document on testing of aquatic toxicity of nano-ZnO in the Framework of the Decision of July 9th 2019 on Zinc Oxide (CAS nr 1314-13-2) under article (46)1 of REACH”. Full detail of the tests and results is presented in the extensive testing reports from the test Institute Fraunhofer (Schlich 2022a-d; Vogt 2022a-d).

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ZnO nano

Nano ZnO chronic toxicity

The present review focused notably on these chronic data, which are considered to be of major relevancy for the risk assessment and the related PNECs. Like for the acute toxicity, the data are presented in the attached tables 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 relevant IUCLID sections.

The freshwater chronic dataset covers 2 invertebrates (mollusc and crustacean). The full set of EC10/NOEC values are presented in the attached tables (attached background material).

The marine chronic dataset covers 1 invertebrate (clam) species.

For freshwater, the comparison of the chronic ecotoxicity data obtained within the same studies for the same species/endpoints after exposing the organisms to either the soluble Zn2+ion or the ZnO-NP form show that there is generally no difference in toxicity between the two Zn-forms.

The general observation of similar toxicity by the Zn-ion and the ZnO-NP is most relevant for the discussion on the aquatic PNEC developed for zinc substances; it can be concluded that the available data show that the general Zn-ion based PNEC for freshwater is relevant for the nano-ZnO, too.