Zinc oxide

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

effects on growth of green algae

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 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

2011

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.

Details on test solutions:

The preparation of the test item solutions followed the procedures described in the OECD TG 318. This included weighing 100.0 mg of the test item into a glass beaker of 250 mL volume, pre-wetted with 1 mL of modified growth medium and left in the form of wet paste for 24 hours to insure the proper interaction of the nanomaterial surface with the water. After 24 h of pre-wetting, the resulting wet paste was dispersed in 149 mL of modified growth medium, thus providing a stock dispersion with a nanomaterial concentration of 666.67 mg test item/L. To ensure a homogenous dispersion of the test material in modified growth medium a sonication probe was applied. The test item dispersion by probe sonication was performed.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

The unicellular green alga Desmodesmus subspicatus is a standard species listed in the OECD 201 guidance (2011) and was as test organism representing primary producers in freshwater. Although other green algal species are listed in the OECD 201 guidance, D. subspicatus was the species specifically chosen by the Competent Authority.

Origin: Experimental Phycology and Culture Collection of Algae at the University of Goettingen (EPSAG), Nikolausberger Weg 18, 37073 Goettingen, Germany.

Strain number: 86.81 SAG.

Cultivation: The stock cultures were maintained fulfilling the criteria of the OECD guideline (culture medium recommended by Bringmann und Kühn (1980)). Prior to testing a pre-culture was established in OECD growth medium (as described below) to obtain exponentially growing algae for the test. The culture duration of the pre-cultures was 3 days.

Test type:

other: Static, but stirred

Water media type:

other: Modified sterilised synthetic growth medium (OECD medium) according to OECD 201 (2011) was used as growth medium.

Total exposure duration:

72 h

Hardness:

0.4 mmol/L

Test temperature:

21.5 - 22.0 °C

pH:

7.86 - 8.36

Conductivity:

Ionic strength 159.7 µS/m2

Nominal and measured concentrations:

Total Zn (mg/l): Nominal: Control, 0.403, 1.27, 4.02, 12.7, 40.2
Total Zn (mg/l): measured (Geometric Mean): 0.003, 0.489, 1.26, 3.77, 11.9, 35.3

Dispersed Zn (mg/l): Nominal: Control, 0.403, 1.27, 4.02, 12.7, 40.2
Dispersed (mg/l): measured (Geometric Mean): 0.05, 0.306, 0.532, 0.790, 0.887, 1.23

Dissolved Zn (mg/l): Nominal: Control, 0.403, 1.27, 4.02, 12.7, 40.2
Dissolved Zn (mg/l): measured (Geometric Mean): 0.008, 0.267, 0.550, 0.764, 0.850, 1.24

Key result

Duration:

72 h

Dose descriptor:

EC10

Remarks:

Total fraction (non-filtered)

Effect conc.:

0.45 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (total fraction)

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Remarks:

Nominal

Effect conc.:

0.42 mg/L

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Remarks:

Dispersed fraction (0.2 µm filter)

Effect conc.:

0.4 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (dissolved fraction)

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Remarks:

Truly dissolved fraction (3 kDa filter)

Effect conc.:

43 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (dissolved fraction)

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Remarks:

Total fraction (non-filtered)

Effect conc.:

3.35 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (total fraction)

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Remarks:

Nominal

Effect conc.:

3.5 mg/L

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Remarks:

Dispersed fraction (0.2 µm filter)

Effect conc.:

0.69 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (dissolved fraction)

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Remarks:

Truly dissolved fraction (3 kDa filter)

Effect conc.:

0.69 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (dissolved fraction)

Basis for effect:

growth rate

Reported statistics and error estimates:

Biological data were statistically analysed to determine EC50 and EC10 values together with 95 % confidence intervals, where possible.
For the total fraction, for growth rate and yield the data was analysed using non- linear regression procedures (3 – parametric normal).
For the dispersed and dissolved fraction, for growth rate there was a lack of fit for the tested 3-parametric non-linear regression procedures (3-parametric normal, logistic and Weibull) and therefore, the ErC50 was calculated based on linear regression procedures (Probit). The yield data was analysed using non- linear regression procedures (3 – parametric Weibull).

The NOEC for growth rate and yield (all fractions) was determined using the Step-down Jonckheere-Terpstra Test Procedure (significance level of 0.05, one-sided smaller).

The computer program ToxRat® was used for statistical evaluations.

To check that the nano material did not reduce the light intensity - the light intensity was checked in the incubation chamber and it ranged from 91.98 to 97.45 µE m^-2 s^-1. In addition, the light intensity underneath a representative replicate of each treatment and the control was measured daily. At test initiation, the light intensity decreased with increasing test item concentration (71.83 – 84.96 µE m^-2 s^-1) compared to the control (87.29 µE m^-2 s^-1). This effect became less pronounced as the test continued. At test termination, the measured light intensity below a control replicate was at 86.97 µE m^-2 s^-1 and ranged between 79.02 and 85.76 µE m^-2 s^-1 in the five treatments.

Validity criteria fulfilled:

yes

Remarks:

In control cultures: the increase in cell numbers, the mean of the replicate coefficients of variations in growth rate and the coefficient of variation of average specific growth rate = all met the test validity criteria

Conclusions:

Based on the evaluation performed on geometric mean concentrations of the total zinc fraction: EC10 (chronic): an EC10 of 0.42 mg nominal Zn/L and 0.45 mg total Zn/L.
EC50 (acute): an EC50 of 3.50 mg nominal Zn/L, and 3.35 mg total Zn/L.

Executive summary:

Exponentially-growing cultures of the green alga were exposed to five concentrations of the test item plus a control over several generations under defined conditions for 72 hours according to the OECD guideline 201 (2011).

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 0.45 and the 'total EC50' value of 3.35 mg/L.

Reference 2

Endpoint:

effects on growth of green algae

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 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

2011

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.

Details on test solutions:

The preparation of the test item solutions followed the procedures described in the OECD TG 318. This included weighing 100.0 mg of the test item into a glass beaker of 250 mL volume, pre-wetted with 1 mL of modified growth medium and left in the form of wet paste for 24 hours to insure the proper interaction of the nanomaterial surface with the water. After 24 h of pre-wetting, the resulting wet paste was dispersed in 149 mL of modified growth medium, thus providing a stock dispersion with a nanomaterial concentration of 666.67 mg test item/L. To ensure a homogenous dispersion of the test material in modified growth medium a sonication probe was applied. The test item dispersion by probe sonication was performed.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

The unicellular green alga Desmodesmus subspicatus is a standard species listed in the OECD 201 guidance (2011) and was as test organism representing primary producers in freshwater. Although other green algal species are listed in the OECD 201 guidance, D. subspicatus was the species specifically chosen by the Competent Authority.

Origin: Experimental Phycology and Culture Collection of Algae at the University of Goettingen (EPSAG), Nikolausberger Weg 18, 37073 Goettingen, Germany.

Strain number: 86.81 SAG.

Cultivation: The stock cultures were maintained fulfilling the criteria of the OECD guideline (culture medium recommended by Bringmann und Kühn (1980)). Prior to testing a pre-culture was established in OECD growth medium (as described below) to obtain exponentially growing algae for the test. The culture duration of the pre-cultures was 3 days.

Test type:

other: Static, but stirred

Water media type:

other: Modified sterilised synthetic growth medium (OECD medium) according to OECD 201 (2011) was used as growth medium.

Total exposure duration:

72 h

Hardness:

0.4 mmol/L

Test temperature:

21.5 - 22.0 °C

pH:

7.86 - 8.36

Conductivity:

Ionic strength 164.9 µS/m2

Nominal and measured concentrations:

Total Zn (mg/l): Nominal: Control, 0.403, 1.27, 4.02, 12.7, 40.2
Total Zn (mg/l): measured (Geometric Mean): 0.008, 0.431, 1.16, 3.79, 12.6, 36.9

Dispersed Zn (mg/l): Nominal: Control, 0.403, 1.27, 4.02, 12.7, 40.2
Dispersed (mg/l): measured (Geometric Mean): 0.015, 0.314, 0.551, 0.832, 1.11, 2.34

Dissolved Zn (mg/l): Nominal: Control, 0.403, 1.27, 4.02, 12.7, 40.2
Dissolved Zn (mg/l): measured (Geometric Mean): 0.015, 0.304, 0.536, 0.814, 1.06, 1.48

Key result

Duration:

72 h

Dose descriptor:

EC10

Remarks:

Total fraction (unfiltered)

Effect conc.:

1.08 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (total fraction)

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Remarks:

Nominal

Effect conc.:

1.16 mg/L

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Remarks:

Dispersed fraction (0.2 µm filter)

Effect conc.:

0.51 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (dissolved fraction)

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Remarks:

Truly dissolved fraction (3 kDa filter)

Effect conc.:

0.56 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (dissolved fraction)

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Remarks:

Total fraction (unfiltered)

Effect conc.:

7.25 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (total fraction)

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Remarks:

Nominal

Effect conc.:

7.6 mg/L

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Remarks:

Dispersed fraction (0.2 µm filter)

Effect conc.:

1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (dissolved fraction)

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Remarks:

Truly dissolved fraction (3 kDa filter)

Effect conc.:

0.92 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

element (dissolved fraction)

Basis for effect:

growth rate

Reported statistics and error estimates:

Biological data were statistically analysed to determine EC50 and EC10 values together with 95 % confidence intervals, where possible.
For the total fraction, for growth rate and yield the data was analysed using non- linear regression procedures (3 – parametric normal).
For the dispersed and dissolved fraction, for growth rate there was a lack of fit for the tested 3-parametric non-linear regression procedures (3-parametric normal, logistic and Weibull) and therefore, the ErC50 was calculated based on linear regression procedures (Probit). The yield data was analysed using non- linear regression procedures (3 – parametric Weibull).

The NOEC for growth rate and yield (all fractions) was determined using the Step-down Jonckheere-Terpstra Test Procedure (significance level of 0.05, one-sided smaller). The computer program ToxRat® was used for statistical evaluations.

To check that the nano material did not reduce the light intensity - the light intensity was checked in the incubation chamber and it ranged from 97.79 to 98.07 µE m^-2 s^-1. In addition, the light intensity underneath a representative replicate of each treatment and the control was measured daily. At test initiation, the light intensity decreased with increasing test item concentration (59.98 - 83.82 µE m^-2 s^-1) compared to the control (88.94 µE m^-2 s^-1). This effect became less pronounced as the test continued. At test termination, the measured light intensity below a control replicate was at 86.21 µE m^-2 s^-1 and ranged between 78.92 and 85.88 µE m^-2 s^-1 in the five treatments.

Validity criteria fulfilled:

yes

Remarks:

In control cultures: the increase in cell numbers, the mean of the replicate coefficients of variations in growth rate and the coefficient of variation of average specific growth rate = all met the test validity criteria

Conclusions:

Based on the evaluation performed on geometric mean concentrations of the total zinc fraction: EC10 (chronic): an EC10 of 1.16 mg nominal Zn/L and 1.08 mg total Zn/L.
EC50 (acute): an EC50 of 7.60 mg nominal Zn/L, and 7.25 mg total Zn/L.

Executive summary:

Exponentially-growing cultures of the green alga were exposed to five concentrations of the test item plus a control over several generations under defined conditions for 72 hours according to the OECD guideline 201 (2011).

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 1.08 and the 'total EC50' value of 7.25 mg/L.

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).

_________________________________________________________________

ZnO nano evaluation

Three Freshwater Alga, Growth Inhibition Tests (OECD 201) were done as part of the nano evaluation testing program in order to assess the effects of nano ZnO on the growth of Desmodesmus subspicatus. Three different nanomaterials were tested: T0368/01, T0410/01 and T0369/01 (hydrophobic surface treated). The data newly generated on D. subspicatus did not show evidence for additional toxicity of ZnO-nanoforms tested.

Nano ZnO acute

The present short-term acute aquatic toxicity database for nano-ZnO, covers 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 attached tables, 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 relevant IUCLID sections. The full set of EC50 values are presented in the tables attached as background material.

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 algae and 1 sediment crustacean. The full set of EC10/NOEC values are presented in the tables attached as background documents.

The marine chronic dataset covers 2 algae 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.

While the data for the freshwater algae do not suggest a higher toxicity of the NP form, the marine algae data set shows a diatom where the Zn2+/ZnO-NP toxicity ratio is higher than 2. A similar observation was made on the acute dataset. Consequently, this observation for the marine risk assessment merits further consideration.