Zinc oxide

Toxicity to terrestrial arthropods

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to terrestrial arthropods: long-term

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

ISO 11267 (Inhibition of Reproduction of Collembola by Soil Pollutants)

GLP compliance:

not specified

Remarks:

GLP compliance not specified in publication

Application method:

soil

Specific details on test material used for the study:

ZnO-NP powder, with a reported diameter size of <200 nm, was purchased from BASF (Z-COTE). Powders were coated with carbon and photos of the particles were taken using a (field emission) scanning electron microscope (JEOL JSM-6301F). Nonnano ZnO (Merck, pro analysi, >99%) and ZnCl2 (Merck, zinc chloride pure) were used for comparison. The diameter size of ZnO-NP powder is at the nano-scale (i.e. <100 nm), although the fraction <100 nm has not been established. The diameter size of non-nano ZnO powder is >200 nm in all cases, and therefore provides a good reference substance to study size-related effects of ZnO.

Analytical monitoring:

yes

Details on sampling:

Soil samples were dried for 24 h at 60 C. For total zinc analysis, 100 mg of dried soil (two replicates per treatment) was digested in a mixture of Milli-Q, concentrated HCl and concentrated HNO3 (1:1:4 by vol.). Tightly closed bombs were placed in an oven (CEM MDS 81-D) at 140 C for 7 h. After digestion, the solution was analysed by flame Atomic Absorption Spectrometry (AAS) (Perkin Elmer 1100B). Certified reference material (ISE sample 989 of River Clay fromWageningen, The Netherlands) was used to ensure the accuracy of the analytical procedure. Measured zinc concentrations in the reference material were within 10% of the certified concentrations.
At the end of the toxicity tests, soil porewater was collected by centrifuging 50 g soil (Centrifuge Falcon 6/300 series, CFC Free), after saturation with Milli-Q and two weeks equilibrium time. Soils were centrifuged for 50 min with a relative force of 2000 g over a round filter (28 mm) and a membrane filter (Whatman filter paper 0.45 mm), placed inside the tubes (method described by Hobbelen et al., 2004).
Approximately 10 ml soil pore water per sample was collected for analysis by flame AAS (Perkin Elmer 1100B). Zinc concentrations in the samples were also determined by flame AAS after ultrafiltration of the soil pore water to obtain a particle-free extract. Soil solutions were centrifuged in a 100 kDa ultrafiltration device (Amicon Ultra-15 Filters, Millipore) for 20 min at 2000 g.
The pH of the soil pore water was measured before and after ultrafiltration using a Consort P907 meter.

Vehicle:

no

Details on preparation and application of test substrate:

Loamy sand soil (LUFA-Speyer 2.2, Sp 2121, Germany, 2009) with a pHCaCl2 of 5.5, a total organic carbon content of 2.09%, a cation exchange capacity (CEC) of 10.0 meq/100 g and a water-holding capacity (WHC) of 46.5% was used. The soil was oven-dried at 60 C overnight prior to the experiment to eliminate undesired soil fauna. The test consisted of seven ZnO-NP concentrations (nominal range 100e6400 mg Zn/kg d.w.), five non-nano ZnO concentrations (400e6400 mg Zn/ kg d.w.), five ZnCl2 concentrations (100e1600 mg Zn/kg d.w.) and a control without zinc. Slightly different exposure scales were chosen for ZnO-NP, non-nano ZnO and ZnCl2, because it is expected that soluble zinc compounds will be more toxic to soil
organisms than insoluble zinc compounds, at least shortly after the addition to the soil. To achieve an as homogeneous distribution as possible, the test compounds were introduced into the soil as aqueous solutions prepared in soil extracts. For that purpose soilewater suspensions
were prepared by mixing air-dried soil with Milli-Q using a soil-water ratio of 2:5 (w/v). The suspensions were shaken at 180 rpm at ambient temperature for 1 h. Soil extracts were filtered under vacuum (Whatman filter paper, type 595) and ZnO-NP, non-nano ZnO and ZnCl2 were added to the filtrates. The solutions, which showed a milky-white colour, were shaken for two days at 180 rpm and carefully mixed with 200 g dry soil. Additional Milli-Q was added to achieve a soil moisture content of 23.3% (w/w) corresponding with 50% of the maximum WHC. Soils were equilibratedfor one day before use in the toxicity test. To visualize the size and degree of aggregation of the ZnO particles, photos of ZnO-NP and non-nano ZnO in the spiking solution (approx. 1000 mg Zn/l) were taken using transmission electron microscope (TEM) operating at 60 kV (JEOL 1010, containing a 2 k CCD camera). Soil solution without ZnO particles was taken for comparison. Samples were dropped on a 75 mesh copper Formvar grid and left to dry before examination by TEM.

Test organisms (species):

Folsomia sp.

Animal group:

Collembola (soil-dwelling springtail)

Details on test organisms:

The springtail F. candida (“Berlin strain”; VU University Amsterdam) was cultured in plastic containers with a moist bottom of plaster of Paris containing 10% charcoal, at 20 1 C at a light/dark regime of 12/12 h. The experiment was initiated with juveniles of the same age (10e12 days) that were obtained by synchronising the egg laying of the culture animals, fed with dried baker’s yeast (Dr. Oetker).

Study type:

laboratory study

Limit test:

no

Total exposure duration:

28 d

Test temperature:

20°C

Reference substance (positive control):

no

Duration:

28 d

Dose descriptor:

LC50

Effect conc.:

> 3 086 mg/kg soil dw

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

Zn from nano ZnO

Basis for effect:

mortality

Duration:

28 d

Dose descriptor:

LC50

Effect conc.:

> 6 282 mg/kg soil dw

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

Zn from bulk ZnO

Basis for effect:

mortality

Duration:

28 d

Dose descriptor:

LC50

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

Zn from ZnCl2

Basis for effect:

mortality

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

1 964 mg/kg soil dw

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

Zn from nano ZnO

Basis for effect:

reproduction

Remarks on result:

other: concentration measured in the soil

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

1 591 mg/kg soil dw

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

Zn from bulk ZnO

Basis for effect:

reproduction

Remarks on result:

other: concentration measured in the soil

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

298 mg/kg soil dw

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

Zn from ZnCl2

Basis for effect:

reproduction

Remarks on result:

other: concentration measured in the soil

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

10.1 other: mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

Zn from nano ZnO

Basis for effect:

reproduction

Remarks on result:

other: concentration measured in the soil pore water

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

7.94 other: mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

Zn from bulk ZnO

Basis for effect:

reproduction

Remarks on result:

other: concentration measured in the soil pore water

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

16.8 other: mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

Zn from ZnCl2

Basis for effect:

reproduction

Remarks on result:

other: concentration measured in the soil pore water

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

1 678 mg/kg soil dw

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

Zn from nano ZnO

Basis for effect:

reproduction

Remarks on result:

other: concentration measured in the soil

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

1 383 mg/kg soil dw

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

Zn from bulk ZnO

Basis for effect:

reproduction

Remarks on result:

other: concentration measured in the soil

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

108 mg/kg soil dw

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

Zn from ZnCl2

Basis for effect:

reproduction

Remarks on result:

other: concentration measured in the soil

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

9.47 other: mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

Zn from nano ZnO

Basis for effect:

reproduction

Remarks on result:

other: concentration measured in the soil pore water

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

6.85 other: mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

Zn from bulk ZnO

Basis for effect:

reproduction

Remarks on result:

other: concentration measured in the soil pore water

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

1.73 other: mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

Zn from ZnCl2

Basis for effect:

reproduction

Remarks on result:

other: concentration measured in the soil pore water

Details on results:

Survival of F. candida in soil spiked at concentrations up to 6400 mg Zn/kg d.w. with ZnO-NP and non-nano ZnO was not affected and comparable to that in the control (i.e. >72%). Survival was affected by ZnCl2 with an LC50 of 1000 mg Zn/kg d.w (95% CI 861e1162). Table 1 summarizes the LC50, EC50 and EC10 values for the effect on F. candida exposed to soil spiked with ZnO-NP, nonnano ZnO and ZnCl2.
The average number of juveniles in the controls was 140 with a coefficient of variance of 40%. Reproduction was reduced in
a dose-dependent manner (Fig. 4) and EC50 values of 1964, 1591 and 298 mg Zn/kg d.w. were estimated for ZnO-NP, non-nano ZnO
and ZnCl2, respectively. According to a likelihood-ratio test, the EC50 of ZnCl2was significantly lower than the EC50s of ZnO-NP and
non-nano ZnO, (c2 (1) ¼ 12.1 and 22.9, p < 0.001), while the EC50s of ZnO-NP and non-nano ZnO were not significantly different
(c2 (1) ¼ 0.67). EC50 values of 10.1, 7.94 and 16.8 mg Zn/l were calculated for ZnO-NP, non-nano ZnO and ZnCl2, respectively based
on measured concentrations in the soil pore water. The pore water EC50 for non-nano ZnO was significantly lower than the one for of
ZnO-NP (c2 (1) ¼ 5.02, p < 0.05). Based on the concentration in the soil pore water, the EC10 of ZnCl2 was 1.73 mg Zn/l, which was
significantly lower value than the EC10 for ZnO-NP (c2 (1) ¼ 28.3, p < 0.05).

Validity criteria fulfilled:

yes

Conclusions:

Very good quality study.

Executive summary:

Effects on springtail reproduction was not related to ZnO particle size and most probably resulted from zinc dissolved from ZnO-NP, non-nano ZnO and ZnCl2 (EC50s from ZnO bulk and nZnO are comparable).

Reference 2

Endpoint:

toxicity to terrestrial arthropods: long-term

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

ISO 11267 (Inhibition of Reproduction of Collembola by Soil Pollutants)

GLP compliance:

not specified

Remarks:

GLP compliance not specified in publication

Application method:

soil

Specific details on test material used for the study:

ZnO nanoparticle powders (Nanosun Zinc Oxide P99/30) with a reported diameter size of 30nm were tested. Transmission Electron Micrographs and Particle Size Distribution of the ZnO nanoparticles are shown by Waalewijn-Kool et al. (2012). Primary particle size of the nanoparticle powder was in agreement with the size reported by the manufacturer. The effect of dissolved Zn was investigated by running tests with the soluble salt ZnCl2. (Merck, zinc chloride pure).

Analytical monitoring:

yes

Vehicle:

no

Test organisms (species):

Folsomia candida

Animal group:

Collembola (soil-dwelling springtail)

Details on test organisms:

The springtail F. candida (Berlin strain, VU University Amsterdam) was cultured in pots with a base of moist plaster of Paris mixed with charcoal at 201 8C at a light/dark regime of 12/12 h. Experiments were initiated with juveniles of the same age (10–12 d) that were obtained by synchronizing the egg laying of the culture animals, fed with dried baker’s yeast (Dr. Oetker).

Study type:

laboratory study

Limit test:

no

Total exposure duration:

28 d

Test temperature:

20+/-1°C

pH (if soil or dung study):

4.5, 5.9 and 7.2 were tested

Photoperiod and lighting:

12/12 photoperiod

Nominal and measured concentrations:

100, 200, 400, 800, 1600, 3200, 6400 mg Zn/L

Reference substance (positive control):

no

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

2 962 mg/kg soil dw

Nominal / measured:

meas. (not specified)

Conc. based on:

element (total fraction)

Remarks:

from nano ZnO, 2.37% OM

Basis for effect:

reproduction

Remarks on result:

other: CI95%: 1389-4534

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

3 493 mg/kg soil dw

Nominal / measured:

meas. (not specified)

Conc. based on:

element (total fraction)

Remarks:

from nano ZnO, 3.09% OM

Basis for effect:

reproduction

Remarks on result:

other: CI95%: 358-6628

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

4 446 mg/kg soil dw

Nominal / measured:

meas. (not specified)

Conc. based on:

element (total fraction)

Remarks:

from nano ZnO, 10.6 OM

Basis for effect:

reproduction

Remarks on result:

other: CI95%: 2830-6061

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

1 695 mg/kg soil dw

Nominal / measured:

meas. (not specified)

Conc. based on:

element (total fraction)

Remarks:

from nano ZnO, 14.7% OM

Basis for effect:

reproduction

Remarks on result:

other: CI95%: 784-2605

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

356 mg/kg soil dw

Nominal / measured:

meas. (not specified)

Conc. based on:

element (total fraction)

Remarks:

from ZnCl2, 2.37% OM

Basis for effect:

reproduction

Remarks on result:

other: CI95%: 24-5378

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

439 mg/kg soil dw

Nominal / measured:

meas. (not specified)

Conc. based on:

element (total fraction)

Remarks:

from ZnCl2, 3.09% OM

Basis for effect:

reproduction

Remarks on result:

other: CI95%: 316-561

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

1 433 mg/kg soil dw

Nominal / measured:

meas. (not specified)

Conc. based on:

element (total fraction)

Remarks:

from ZnCl2, 10.6% OM

Basis for effect:

reproduction

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

1 695 mg/kg soil dw

Nominal / measured:

meas. (not specified)

Conc. based on:

element (total fraction)

Remarks:

from ZnCl2, 14.7% OM

Basis for effect:

reproduction

Reported statistics and error estimates:

EC50 values for the effect on reproduction were estimated applying the logistic model of Haanstra et al.(1985) and were determined based on total Zn concentrations in soil and dissolved Zn and free Zn2+ concentrations in the porewater. A generalized likelihood ratio test (Sokal and Rohlf, 1995) was applied to compare EC50 values based on total Zn concentration obtained for both Zn forms and for each soil.Calculations wereperformed in SPSS Statistics 20.

Validity criteria fulfilled:

yes

Conclusions:

Reproduction was reduced in a dose response manner for both zinc forms in all 4 soils. EC50 of ZnCl2 increased with increasing OM content but no significant relation of EC50 with soil OM content was seen for nano ZnO. Highest toxicity was observed in the most organic soil but its EC50 value was not significantly lower than the ones for the less organic soils. EC50 value of ZnCl2 was significantly lower than EC50 nano ZnO in soil 1 and 2 (lowest OM content)

Executive summary:

The toxicity of ZnCl2 was related to soil OM content but this was not the case for ZnO–NP, suggesting that ZnO–NP have to dissolve to make Zn bioavailable.Soil pH and DOC play an important role in the dissolution of ZnO–NP and its release into the pore water. Further research is necessary on interactions between ZnO–NP and soil components such as OM, affecting ZnO–NP toxicity but it cannot be seen separately from the role of other soil properties, such as pH.

Reference 3

Endpoint:

toxicity to terrestrial arthropods: long-term

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

ISO 11267 (Inhibition of Reproduction of Collembola by Soil Pollutants)

GLP compliance:

not specified

Remarks:

GLP compliance not specified in publication

Application method:

soil

Specific details on test material used for the study:

Two types of ZnO with different sizes were applied in the toxicity tests: 30 nm ZnO nanoparticles (Nanosun Zinc Oxide P99/30) and 200 nm non-nano ZnO (Microsun Zinc Oxide W45/30).

Analytical monitoring:

yes

Vehicle:

no

Test organisms (species):

Folsomia candida

Animal group:

Collembola (soil-dwelling springtail)

Details on test organisms:

The springtail F. candida (Berlin strain, VU University Amsterdam) was cultured in pots with a base of moist plaster of Paris mixed with charcoal at 201 8C at a light/dark regime of 12/12 h. Experiments were initiated with juveniles of the same age (10–12 d) that were obtained by synchronizing the egg laying of the culture animals, fed with dried baker’s yeast (Dr. Oetker).

Study type:

laboratory study

Limit test:

no

Total exposure duration:

28 d

Test temperature:

20+/-1°C

pH (if soil or dung study):

4.5, 5.9 and 7.2 were tested

Photoperiod and lighting:

12/12 photoperiod

Nominal and measured concentrations:

100, 200, 400, 800, 1600, 3200, 6400 mg Zn/L

Reference substance (positive control):

no

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

553 mg/kg soil dw

Nominal / measured:

estimated

Conc. based on:

element (total fraction)

Remarks:

from nano ZnO, pH 4.5

Basis for effect:

reproduction

Remarks on result:

other: CI95%:208-896

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

331 mg/kg soil dw

Nominal / measured:

estimated

Conc. based on:

element (total fraction)

Remarks:

from ZnCl2, pH 4.5

Basis for effect:

reproduction

Remarks on result:

other: CI95%: 264-399

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

1 481 mg/kg soil dw

Nominal / measured:

estimated

Conc. based on:

element (total fraction)

Remarks:

from nano ZnO, pH 5.9

Basis for effect:

reproduction

Remarks on result:

other: CI95%: 1204-1758

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

732 mg/kg soil dw

Nominal / measured:

estimated

Conc. based on:

element (total fraction)

Remarks:

from ZnCl2, pH 5.9

Basis for effect:

reproduction

Remarks on result:

other: CI95%: 543-920

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

3 233 mg/kg soil dw

Nominal / measured:

estimated

Conc. based on:

element (total fraction)

Remarks:

from nano ZnO, pH 7.2

Basis for effect:

reproduction

Remarks on result:

other: CI95%: 92.3-6373

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

1 174 mg/kg soil dw

Nominal / measured:

estimated

Conc. based on:

element (total fraction)

Remarks:

from ZnCl2, pH 7.2

Basis for effect:

reproduction

Remarks on result:

other: CI95%: 546-1802

Reported statistics and error estimates:

The trimmed Spearman-Karber method was applied to estimate median lethal concentration (LC50) values for the effect of ZnCl2 on springtail survival [20]. The EC50 values for reproduction were estimated applying the logistic model of Haanstra et al. [21] and determined based on total Zn, dissolved Zn, and free Zn2þ ion concentrations. A generalized likelihood ratio test [22] was applied to compare EC50 values obtained for
each Zn form and each test soil and to compare EC50 values based on dissolved Zn concentrations before and after ultrafiltration. Calculations were performed in SPSS Statistics 20.

Validity criteria fulfilled:

yes

Conclusions:

In the present study we showed that soil pH influences the toxicity of ZnO nanoparticles to F. candida and that ZnO nanoparticles and ZnCl2 were more toxic in acidic soil than in basic soil. In comparison with ZnCl2, ZnO nanoparticle toxicity was lower based on total Zn concentrations.
Study suggests that ZnO nanoparticle toxicity in soil is a Zn speciation problem rather than a particle problem, with soil pH having an important role in ZnO nanoparticle dissolution, bioavailability, and toxicity.

Reference 4

Endpoint:

toxicity to terrestrial arthropods: long-term

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

ISO 11267 (Inhibition of Reproduction of Collembola by Soil Pollutants)

GLP compliance:

not specified

Remarks:

GLP compliance not specified in publication

Application method:

soil

Specific details on test material used for the study:

Two sizes of zinc oxide (ZnO) were applied, namely 30 nm (Nanosun ZnO P99/30) and 200 nm (Microsun ZnO W45/ 30).

Analytical monitoring:

yes

Details on sampling:

Five samples per treatment (±100 mg dried soil) were randomly taken from the batches of spiked soil and digested in a mixture of Milli-Q, concentrated HCl and concentrated HNO3 (1:1:4 by vol.) using an oven (CEM MDS 81-D). After digestion for 7 h at 140 C, solutions were analysed for total zinc concentration by flame atomic absorption spectrometry (AAS) (Perkin-Elmer 1100B). Certified reference material (ISE sample 989 of River Clay
from Wageningen, The Netherlands) was used to ensure the accuracy of the analytical procedure. Measured zinc concentrations in the reference material were within 10 % of the certified concentrations. A two-sided Student t test was performed to compare spiking methods for each concentration.
Soil pore water was collected by centrifuging 50 g soil (Centrifuge Falcon 6/300 series, CFC Free), after saturation with Milli-Q and one week equilibration. Soils were centrifuged for 50 min with a relative force of 2,000 g over a round filter and a membrane filter (Whatman 0.45 lm),
placed inside the tubes (method described by Hobbelen et al. 2004). Approximately 10 ml pore water per sample was collected for analysis by flame AAS (Perkin-Elmer 1100B). Zinc concentrations in the samples were also determined by flame AAS after ultrafiltration of the soil pore water to obtain a particle-free extract. For this, soil solutions were centrifuged in a 100 kDa ultrafiltration device (Amicon Ultra-15 Filters, Millipore) for 20 min at
2,000 g.

Vehicle:

no

Test organisms (species):

Folsomia candida

Animal group:

Collembola (soil-dwelling springtail)

Details on test organisms:

Berlin strain’’; VU UniversityAmsterdam

Study type:

laboratory study

Limit test:

no

Total exposure duration:

28 d

Reference substance (positive control):

no

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

3 159 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

element

Remarks:

Zn

Basis for effect:

reproduction

Remarks on result:

other: 30 nm spiked in soil as dry powder

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

2 914 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

element

Remarks:

Zn

Basis for effect:

reproduction

Remarks on result:

other: 200 nmspiked in soil as dry powder

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

3 593 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

element

Remarks:

Zn

Basis for effect:

reproduction

Remarks on result:

other: 30 nm spiked in soil as suspension

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

5 633 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

element

Remarks:

Zn

Basis for effect:

reproduction

Remarks on result:

other: 200 nm spiked in soil as suspension

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

2 559 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

element

Remarks:

Zn

Basis for effect:

reproduction

Remarks on result:

other: 30 nmspiked in soil as dry powder

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

2 730 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

element

Remarks:

Zn

Basis for effect:

reproduction

Remarks on result:

other: 200 nm spiked in soil as dry powder

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

3 611 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

element

Remarks:

Zn

Basis for effect:

reproduction

Remarks on result:

other: 200 nm spiked in soil as suspension

Survival of F. candida in soil spiked at concentrations up to 6,400 mg Zn kg-1 was not affected for both ZnO powders. The average numbers of juveniles in the two control soils were 206 (±91.7, n = 5) for dry spiking series and 81 (±45.1, n = 5) for suspension spiking. The effect of 30 and 200 nm ZnO particles on reproduction was concentration-dependent with a steeper dose– response curve for dry spiking than for suspension spiking. The EC50 values for the effect on the reproduction of F. candida of 30 and 200 nm ZnO were 3,159 (95 % confidence interval: 126–5,502) and 2,914 (1,813– 6,787) mg Zn kg-1 for the dry powder series. For the suspension- spiked soils, the EC50s were 3,593 (122–5,684) and 5,633 (3,711–5,608) mg Zn kg-1 for 30 and 200 nm ZnO.

Large 95 % confidence intervals were estimated and no significant differences between the EC50 values were found when applying a generalized likelihood-ratio test (v2\3.84; n.s.). Corresponding EC10 values were 2,559 (133–5,502) mg Zn kg-1 for the 30 nm ZnO particles and 2,730 (149–6,787) for the 200 nm particles spiked in the soil as dry powder. For the 200 nm particles spiked as suspension EC10 was 3,611 (121–5,608) mg Zn kg-1. No EC10 could be calculated for the 30 nm ZnO particles spiked as suspension. EC50 values based on porewater concentrations could not be estimated due to the small differences in zinc

concentrations measured in the pore water.

Validity criteria fulfilled:

yes

Conclusions:

Good quality study and considered useful for consideration

Executive summary:

Folsomia candida organisms were exposed during 28d at nZnO particles. ZnO-NP as such may have an effect on F. candida at concentrations between 1600 and 6400 mg Zn/kg. This study shows that both 30 and 200 nm ZnO particles were equally toxic to F. candida after 28days exposure in natural soil.

Reference 5

Endpoint:

toxicity to terrestrial arthropods: short-term

Type of information:

experimental study

Adequacy of study:

disregarded due to major methodological deficiencies

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: Documentation insufficient for assessment. Original reference in foreign language (Chinese)

Principles of method if other than guideline:

In comparison to two actives of insecticides (cypermethrin and alpha-terthienyl) on their own and these actives mixed with ordinary sized zinc and copper oxides, the synergistic actions for mixtures, cypermethrin and alpha-terthienyl with nano-sized zinc oxide and copper oxide, on pear rust mites were evaluated.

GLP compliance:

no

Application method:

other: dipping

Specific details on test material used for the study:

- Name of test material (as cited in study report): nano zinc oxide
- Analytical purity: 99.9%
- Median size: 30 nm
-Range of particle size: ≤ 80 nm)

Further test substances:
- Nano copper oxide (median particle size: 25 nm, range of particle size: ≤ 60 nm, analytical purity: 99.9%)
- Zinc oxide (range of particle size: 45 - 75 µm, analytical purity: ≥ 99.0%)
- Copper oxide (range of particle size: 150 µm, analytical purity: ≥ 99.0%)
- Cypermethrin (99%, Jinagmeng Pesticide, China)
- Alphaterthienyl (sigma)

Analytical monitoring:

no

Vehicle:

yes

Details on preparation and application of test substrate:

- Test medium preparation: The actives were dissolved in a small amount of acetone (≤ 1%) and emulsified with 5 - 10% of polysorbate-80. Following this, serial dilutions of the test media were prepared with deionised water.
- Controls: 1% acetone
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): 1% acetone as vehicle and 5 - 10% polysorbate 80 as emulsfier
- Concentration of vehicle in test medium (stock solution and final test solution): No data
- Evaporation of vehicle before use: No data

Test organisms (species):

other: Epitrimerus pyri

Animal group:

Acari (leaf-dwelling predatory mite)

Details on test organisms:

TEST ORGANISM
- Common name: Pear rust mite
- Source: Leaves of green jujube (South China Agricultural University)
- Date of collection: no data
- Cultural background: No application of any pesticides for 6 months.
- Disease free: no data

Study type:

laboratory study

Total exposure duration:

48 h

Remarks:

After the treatments with the test media for 5 seconds, the leaves with mites were incubated for 24 and 48 hours.

Post exposure observation period:

24 or 48 hours

Test temperature:

25 ± 1 °C

Humidity:

80%

Details on test conditions:

TEST SYSTEM
- Test container / cage (material, size): Petri dishes (9 cm)
- No. of replicates per treatment group: 3
- No. of replicates per per control / vehicle control: 3


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Mortality


VEHICLE CONTROL PERFORMED: yes


TEST CONCENTRATIONS
- Spacing factor for test concentrations: No data
- Range finding study
- Test concentrations: The data were not given in the publication

Nominal and measured concentrations:

not given in the publication.

Reference substance (positive control):

no

Duration:

24 h

Dose descriptor:

LC50

Effect conc.:

445.6 other: mg/L

Basis for effect:

mortality

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

374.34 other: mg/L

Basis for effect:

mortality

Duration:

24 h

Dose descriptor:

LC50

Effect conc.:

38.83 other: mg/L

Basis for effect:

mortality

Remarks on result:

other: mixture of nano-zinc oxide and cypermethrin (1:4)

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

14.18 other: mg/L

Basis for effect:

mortality

Remarks on result:

other: mixture of nano-zinc oxide and cypermethrin (1:4)

Duration:

24 h

Dose descriptor:

LC50

Effect conc.:

11.44 other: mg/L

Basis for effect:

mortality

Remarks on result:

other: mixture of nano-zinc oxide and alpha-terthienyl (1:4)

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

9.22 other: mg/L

Basis for effect:

mortality

Remarks on result:

other: mixture of nano-zinc oxide and alpha-terthienyl (1:4)

According to the authors the actives Cypermethrin and alpha Terhienyl mixed with nano-particled zinc oxide and copper oxide showed syntergistic effects to the tested mite.

Test substance	24 hours		48 hours
	LC50 (mg/L)	CTC	LC50 (mg/L)	CTC
Nano-Zinc oxide	445.60		374.34
Nano-copper oxide	866.55		478.65
Cypermethrin	63.80		42.20
Alpha-terthienyl	13.02		5.16
Nano-Zinc oxide + cypermethrin (1:4)	38.83	198.28	14.18	403.96
Nano-Copper oxide + cypermethrin (1:4)	55.93	140.01	20.46	281.42
Nano-Zinc oxide + alpha-terthienyl (1:4)	11.44	145.54	9.22	69.75

Validity criteria fulfilled:

not specified

Reference 6

Endpoint:

toxicity to terrestrial arthropods: long-term

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:

according to guideline

Guideline:

ISO 11267 (Inhibition of Reproduction of Collembola by Soil Pollutants)

GLP compliance:

not specified

Remarks:

GLP compliance not specified in publication

Application method:

soil

Specific details on test material used for the study:

ZnO nanopowder was purchased from Sigma-Aldrich (CAS Number: 1314-13-2), with a nominal primary particle size of less than 100 nm (i.e., rp≤50 nm).

Analytical monitoring:

yes

Vehicle:

no

Details on preparation and application of test substrate:

According to the OECD guidelines, standard artificial soil, composed by sphagnum peat (10%), kaolin clay (20%) and quartz sand (70%), was used both as control soil and as matrix for Zn contamination.

Test organisms (species):

Folsomia candida

Animal group:

Collembola (soil-dwelling springtail)

Study type:

laboratory study

Limit test:

no

Total exposure duration:

28 d

Test temperature:

20±2°C

Reference substance (positive control):

no

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

230 mg/kg soil dw

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Remarks:

Zn

Basis for effect:

reproduction

Details on results:

Zinc seemed to exhibit no adverse effect upon collembolan reproduction at the tested concentration (230 mg kg−1) regardless of the form. In contrast, both ZnO NPs and ZnCl2 spiked soils produce a clear biostimulation (106% and 94%) with respect to the control (OECD soil).

However, in short time tests with collembolans (avoidance test), ZnO NP spiked soil produced only a 16% avoidance during the 100-min observation time with respect to a 76% avoidance for ZnCl2 probably due to oral uptake.

Validity criteria fulfilled:

yes

Conclusions:

Good quality study and considered to be useful for assessing terrestrial toxicity

Executive summary:

Zinc seemed to exhibit no adverse effect upon collembolan reproduction at the tested concentration (230 mg kg−1) regardless of the form. In contrast, both ZnO NPs and ZnCl2 spiked soils produce a clear biostimulation (106% and 94%) with respect to the control (OECD soil).

Description of key information

The toxicity of the nano-ZnO form is in general lower than the toxicity of the zinc ion. Ingestion of solids is a potential risk pathway of greater importance for organisms in soils/sediments than in aquatic systems – potentially a greater chance for exposure to dispersed/aggregated MNPs, and nano particles can be more stable due to e.g. coating or given environmental conditions. However, there is no indication of toxicity deviating from the zinc-ion in the present dataset.

Additional information

There are data on the effect of nano-ZnO (compared to zinc ion toxicity) available for soil invertebrates (4 species), plants (10 species) and soil microorganisms. These data are summarized in the attached table in the attached background material section, where the effect concentrations observed for the Zn2+form are normalized (100%).

From these data, it follows that the toxicity of the nano-ZnO form is in general lower than the toxicity of the zinc ion. Ingestion of solids is a potential risk pathway of greater importance for organisms in soils/sediments than in aquatic systems – potentially a greater chance for exposure to dispersed/aggregated MNPs, and nano particles can be more stable due to e.g. coating or given environmental conditions. However, there is no indication of toxicity deviating from the zinc-ion in the present dataset.