Zinc oxide

Toxicity to terrestrial plants

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to terrestrial plants: long-term

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Good quality study, however no guideline followed. Only LOECs available.

Qualifier:

no guideline followed

Principles of method if other than guideline:

Germination percentage, number of leaves, and primary root elongation, were measured 18 d after planting, toward the full mature vegetative growth stage of the A. thaliana life cycle.

GLP compliance:

not specified

Remarks:

GLP compliance not specified in publication

Specific details on test material used for the study:

The ZnO NPs and microparticles (MPs) were purchased from Sigma Aldrich Chemical Co. (St. Louis, MO, USA). In nutrition solution state, individual of ZnO NPs are rod and nonspherical in shape, and ZnO MPs are also nonspherical particles with little extent of aggregation than that of NPs. The particle size of ZnO NPs was 44.46±4.84 nm and ZnO MPs's particle size was 2~5 μm, which were almost the same as that (ZnO NPs, <50 nm; ZnO MPs, <5 μm) from producer. ZnO NPs purity was >97 % and the specific surface area was >10.8 m2 g−1. ZnO MPs had a purity of 99 % and a specific surface area of 3–4 m2 g−1.

Analytical monitoring:

yes

Vehicle:

no

Species:

other: Arabidopsis thaliana

Plant group:

Dicotyledonae (dicots)

Details on test organisms:

Wild-type Arabidopsis thaliana, Col-0 seeds were purchased from Arabidopsis Biological Resource Center, Ohio State University and stored in a dry opaque envelope at room temperature. The seeds were transferred into 2-ml collection tubes, soaked in 1 ml of autoclaved DI water for 30 min, and
centrifuged (9,000 rpm) for 30 s to soften the seed coat. Seeds were sterilized by washing once with 1 ml of 70% ethanol for 1 min, centrifuging for 30 s, once with 1% sodium hypochlorite for 1 min, centrifuging for 30 s, then four times with 1 ml of autoclaved DI water, and centrifuging for 30 s. Prior to transferring to plates for toxicity experiments, the seeds were suspended in autoclaved 0.1% agar solution in collection tubes in a dark container for 5 d at 48°C. All procedures were conducted under a Steriguard1 laminar hood to prevent microbial contamination.

Test type:

seed germination/root elongation toxicity test

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

18 d

Reference substance (positive control):

not specified

Species:

other: Arabidopsis thaliana

Duration:

18 d

Dose descriptor:

LOEC

Effect conc.:

400 other: mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

germination

Species:

other: Arabidopsis thaliana

Duration:

18 d

Dose descriptor:

LOEC

Effect conc.:

400 other: mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: root elongation

Species:

other: Arabidospsis thaliana

Duration:

18 d

Dose descriptor:

LOEC

Effect conc.:

400 other: mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: number of leaves

Species:

other: Arabidopsis thaliana

Duration:

18 d

Dose descriptor:

LOEC

Effect conc.:

14.6 other: mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

dissolved

Remarks:

Zn

Basis for effect:

germination

Species:

other: Arabidopsis thaliana

Duration:

18 d

Dose descriptor:

LOEC

Effect conc.:

14.6 other: mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

dissolved

Remarks:

Zn

Basis for effect:

other: root elongation

Reported statistics and error estimates:

Each treatment was conducted in at least triplicate, and the results were presented as mean values with respective standard deviations. Phytotoxicity endpoints for all treatments were compared to those of unexposed controls using the Student’s t test paired two sample for means. This test was selected because all seeds came from the same population. Statistical significance of differences between treatments was determined at the 95% confidence level.

ZnO added (mg/L)	nZnO (total dissolved Zn mg/L)	larger ZnO particles (total dissolved Zn mg/L)
0	0.9 +- 0.01	0.9 +- 0.01
400	14.6 +- 0.14	12.89 +- 0.11
4000	96.9 +- 1.22	32.74 +-0.24

Validity criteria fulfilled:

yes

Conclusions:

Good quality study

Executive summary:

Effects of nZnO on Arabidopsis thaliana were seen at concentrations of 400 mg nZnO/L; which releases 14.6 mg Zn/L.

Reference 2

Endpoint:

toxicity to terrestrial plants: 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:

no guideline followed

Principles of method if other than guideline:

Two sets of experiments (24 assays) were performed to evaluate the potential for Zn accumulation and translocation in S. tabernaemontani. Treatment 1 was set up to include 9 mesocosms (with three replicates each) spiked with ZnO NPs at Zn levels of 10, 100 and 1000 mg/L, respectively. In treatment 2, ZnCl2 was spiked into another 9 vessels (with three replicates each) containing nutrient solutions at the same Zn levels of 10, 100 and 1000 mg/L. Control
tests (3 vessels) were also performed where neither ZnO NPs or Zn ions were spiked into the nutrient solutions Additionally, in order to investigate the contribution of Zn2+ released from ZnO NPs to plant phytotoxicity, a set of 3 mesocosms spiked with Zn2+ at 4.0 mg/L was included in the experimental design, as the equilibrium concentration of dissolved Zn2+ was measured in the ZnO NP suspension (1000 mg/L) to be 3.75 mg/L (see Section 3.1).
Each assay was covered with a plastic sheet with 4 holes through which the plant shoots could extend for exposure to light. The plants were harvested at each of the four exposure periods studied (i.e., 3, 7, 14 and 21 d) for biomass weight and Zn analysis.

GLP compliance:

not specified

Remarks:

GLP compliance not specified in publication

Specific details on test material used for the study:

Zinc sulfate heptahydrate (99.8% purity) and ZnO NP with a nominal primary particle size of 35 ± 5 nm (99.8% purity) were purchased from Sigma–Aldrich (Singapore).

Analytical monitoring:

yes

Details on sampling:

At times of 4, 8, 16, 24 and 72 h, the concentration of dissolved Zn in the suspensions, was determined by Zn elemental analysis via inductively coupled plasma optical emission spectroscopy (ICP-OES, Perkin–Elmer, Optima 2100, USA) after centrifugation (10000 rpm for 30 min).

Vehicle:

no

Species:

other: Schoenoplectus tabernaemontani

Plant group:

Monocotyledonae (monocots)

Details on test organisms:

0.2 m high S. tabernaemontani was purchased from Uvaria Tide (Singapore), and thoroughly washed to remove any soil particles attached to the plants. The plants were then acclimatized to Hoagland nutrient solutions
for four weeks. Then the plants with a uniform size were transferred to 2 L mesocosms (4 plants per mesocosm corresponding to the four exposure periods studied, i.e., 3, 7, 14 and 21 d) which contained the modified Hoagland nutrient solution enriched with either ZnO NPs or Zn2+.

Test type:

other: plant growth

Study type:

laboratory study

Substrate type:

other: Zn spiked Hoagland solution

Limit test:

no

Total exposure duration:

21 d

Test temperature:

30°C

pH:

6.7 +/- 1

Nominal and measured concentrations:

10, 100, 1000 mg/l

Reference substance (positive control):

no

Species:

other: Schoenoplectus tabernaemontani

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

1 000 other: mg/l solution

Nominal / measured:

meas. (not specified)

Conc. based on:

element (dissolved fraction)

Remarks:

from ZnCl2

Basis for effect:

growth

Remarks:

54% inhibition compared to control

Species:

other: Schoenoplectus tabernaemontani

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

1 000 other: mg/l solution

Nominal / measured:

meas. (not specified)

Conc. based on:

element (dissolved fraction)

Remarks:

from nano ZnO

Basis for effect:

growth

Remarks:

41% inhibition compared to control

Reported statistics and error estimates:

The results are presented as mean ± SD (standard deviation) for each concentration. Tests to determine statistical differences between treatments were carried out by comparing the critical value through ANOVA one-way analysis of variance (SPSS Statistics V17.0). Comparisons were considered significantly different at p < 0.05.

S. tabernaemontani showed significant (p < 0.05) inhibition compared to the controls when treated by Zn2+ and ZnO NPs at 1000 mg/L. Zn2+ showed more pronounced effect (the shoots became yellow, withered and dry) of toxicity than did ZnO NPs. After 21-d, at Zn concentrations of 10, 100 and 1000 mg/L, the S. tabernaemontani was inhibited by 9%, 28% and 54% by Zn2+ ions (as compared to controls), while values were 6%, 13% and 41% for ZnO NPs.

Validity criteria fulfilled:

yes

Conclusions:

Phytotoxicity exerted by ZnO NPs and Zn2+ was dependent on the Zn concentration as well as exposure time. ZnCL2 inhibit more growth than ZnO NP.

Reference 3

Endpoint:

toxicity to terrestrial plants: long-term

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:

no guideline followed

GLP compliance:

not specified

Remarks:

GLP compliance not specified in paper

Specific details on test material used for the study:

The ZnO-NP dispersion, synthesized by the hydrolysis of a zinc salt in a polyol medium heated to 160 °C, was purchased from Sigma Aldrich (catalog no. 721077). This product has a reported particle size <100 nm measured by dynamic light scatting (DLS) and an average particle size <35 nm measured using an aerodynamic particle sizer (APS) spectrometer.

Analytical monitoring:

yes

Vehicle:

no

Species:

other: Vigna unguiculata

Plant group:

Dicotyledonae (dicots)

Details on test organisms:

Common name: cowpea
Pearl millet seeds naturally aged for 1 year (season) were procured from the Seed Bank, Central Research Farm, Central Arid Zone Research Institute (CAZRI), Jodhpur, India.

Test type:

early seedling growth toxicity test

Study type:

laboratory study

Substrate type:

other: soultion culture and soil culture

Limit test:

yes

Total exposure duration:

4 wk

Test temperature:

28°C (day) to 20°C (night)

pH:

pH 6.7 (oil) and pH 6.1 (solution)

Nominal and measured concentrations:

Nominal. Solution culture = 25 mg/L ZnO and ZnCl2; Soil culture = 500 mg/Kg soil ZnO and ZnCl2

Reference substance (positive control):

no

Species:

other: Vigna unguiculata

Duration:

4 wk

Dose descriptor:

other: single dose effect concentration

Effect conc.:

25 other: mg/L

Nominal / measured:

nominal

Conc. based on:

element (total fraction)

Remarks:

ZnO nano

Basis for effect:

growth

Remarks:

root biomass, solution culture

Remarks on result:

other: 0.08 g/plant

Remarks:

higher than for ZnCl2

Species:

other: Vigna unguiculata

Duration:

4 wk

Dose descriptor:

other: single dose effect concentration

Effect conc.:

25 other: mg/L

Nominal / measured:

nominal

Conc. based on:

element (total fraction)

Remarks:

ZnCl2

Basis for effect:

growth

Remarks:

root biomass, solution culture

Remarks on result:

other: 0.04 g/plant

Remarks:

lower than for ZnO nano

Species:

other: Vigna unguiculata

Duration:

4 wk

Dose descriptor:

other: single dose effect concentration

Effect conc.:

25 other: mg/L

Nominal / measured:

nominal

Conc. based on:

element (total fraction)

Remarks:

ZnO nano

Basis for effect:

growth

Remarks:

stem biomass, solution culture

Remarks on result:

other: 0.21 g/plant

Remarks:

higher than for ZnCl2

Species:

other: Vigna unguiculata

Duration:

4 wk

Dose descriptor:

other: single dose effect concentration

Effect conc.:

25 other: mg/L

Nominal / measured:

nominal

Conc. based on:

element (total fraction)

Remarks:

ZnCl2

Basis for effect:

growth

Remarks:

stem biomass, solution culture

Remarks on result:

other: 0.13 g/plant

Remarks:

lower than for ZnO nano

Species:

other: Vigna unguiculata

Duration:

4 wk

Dose descriptor:

other: single dose effect concentration

Effect conc.:

25 other: mg/L

Nominal / measured:

nominal

Conc. based on:

element (total fraction)

Remarks:

ZnO nano

Basis for effect:

growth

Remarks:

leaf biomass, solution culture

Remarks on result:

other: 0.08 g/plant

Remarks:

higher than for ZnCl2

Species:

other: Vigna unguiculata

Duration:

4 wk

Dose descriptor:

other: single dose effect concentration

Effect conc.:

25 other: mg/L

Nominal / measured:

nominal

Conc. based on:

element (total fraction)

Remarks:

ZnCl2

Basis for effect:

growth

Remarks:

leaf biomass, solution culture

Remarks on result:

other: 0.04 g/plant

Remarks:

lower than for ZnO nano

Species:

other: Vigna unguiculata

Duration:

4 wk

Dose descriptor:

other: single dose effect concentration

Effect conc.:

500 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

element (total fraction)

Remarks:

ZnO nano

Basis for effect:

growth

Remarks:

root biomass, soil culture

Remarks on result:

other: 0.14 g/plant

Remarks:

not significantly different to ZnCl2

Species:

other: Vigna unguiculata

Duration:

4 wk

Dose descriptor:

other: single dose effect concentration

Effect conc.:

500 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

element (total fraction)

Remarks:

ZnCl2

Basis for effect:

growth

Remarks:

root biomass, soil culture

Remarks on result:

other: 0.14 g/plant

Remarks:

not significantly different to ZnO nano

Species:

other: Vigna unguiculata

Duration:

4 wk

Dose descriptor:

other: single dose effect concentration

Effect conc.:

500 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

element (total fraction)

Remarks:

ZnO nano

Basis for effect:

growth

Remarks:

stem biomass, soil culture

Remarks on result:

other: 0.24 g/plant

Remarks:

not significantly different to ZnCl2

Species:

other: Vigna unguiculata

Duration:

4 wk

Dose descriptor:

other: single dose effect concentration

Effect conc.:

500 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

element (total fraction)

Remarks:

ZnCl2

Basis for effect:

growth

Remarks:

stem biomass, soil culture

Remarks on result:

other: 0.22 g/plant

Remarks:

not significantly different to ZnO nano

Species:

other: Vigna unguiculata

Duration:

4 wk

Dose descriptor:

other: single dose effect concentration

Effect conc.:

500 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

element (total fraction)

Remarks:

ZnCl2

Basis for effect:

growth

Remarks:

leaf biomass, soil culture

Remarks on result:

other: 0.12 g/plant

Remarks:

not significantly different to ZnCl2

Species:

other: Vigna unguiculata

Duration:

4 wk

Dose descriptor:

other: single dose effect concentration

Effect conc.:

500 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

element (total fraction)

Remarks:

ZnCl2

Basis for effect:

growth

Remarks:

leaf biomass, soil culture

Remarks on result:

other: 0.11 g/plant

Remarks:

not significantly different to ZnO nano

Details on results:

Results are presented in the Supplementary Material. In solution culture, the addition of Zn reduced plant growth compared to that in the control (basal nutrient solution), with toxicity more severe in ZnCl2 solutions than with those containing ZnO-NPs. In contrast, there were no significant effects (p > 0.05) on plant growth between the control and the ZnO-NP and ZnCl2 treatments in soil culture.

Reported statistics and error estimates:

Treatment differences were tested for significance (p < 0.05) using a one-way analysis of variance (ANOVA) performed with IBM SPSS Statistics 20.

Validity criteria fulfilled:

yes

Conclusions:

Acceptable study, useful supporting study or assessing terrestrial plant toxicity.

Executive summary:

Zn ion from ZnCl generally had a greater effect on all of the endpoints tested (root, stem and leaf biomass). Significant effects were observed in solution culture (25 mg Zn/L) but not in soil culture (500 mg/kg dw soil). Useful supporting study, shows toxicity to cowpea is dependinant on ion concentration.

Reference 4

Endpoint:

toxicity to terrestrial plants: short-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:

no guideline followed

Principles of method if other than guideline:

The bulbs of garlic were allowed to germinate in DI-water until the radicals had elongated to 2.0 cmin length. The seedlings were selected for uniformity in size and shape. Twenty-five seedlings (five seedlings per treatment) were directly placed in ZnO NPs suspensions (10, 20, 30, 40, 50 mg/L) or Zn2+ reference solutions (0, 0.5, 1, 1.5, 2 mg/L) for 24 h. At the end of each treatment, the seedlings were washed carefully with DI-water, and transferred to beakers with DI-water and allowed to germinate at room temperature of 22 ± 1C. Controlled seedlings were placed in DI-water. The roots were protected from direct sunlight in order to minimize fluctuation of the rate of cell division. After 24 h, the root length of the treated seedlings and the controls were measured.

GLP compliance:

not specified

Remarks:

GLP compliance not specified in publication

Specific details on test material used for the study:

ZnO NPs was prepared. The process was as follows: 0.1 M ethanol solution of LiOH was slowly added to 0.12 M ethanol solution of Zn(CH3COO)2 2H2O kept at 0C. The mixed solution was then hydrolyzed in an ultrasonic bath at about 0C for 25 min before a certain amount of hexane was added into the colloidal solution. White precipitate was obtained, centrifuged and washed with de-ionized water three times to wash out the ethanol, and was then dried in a vacuum condition to obtain ZnO quantum dot powders.

Analytical monitoring:

not specified

Vehicle:

no

Species:

other: Allium sativum

Plant group:

Monocotyledonae (monocots)

Details on test organisms:

Garlic (Allium sativum L., China origin 55/70 mm CAT.1) was purchased from a local market. Healthy and equal-sized garlic cloves were selected. The bulbs had not initiated the formation of root growth. Before commencing the experiment, the dry scales of the bulbs were removed. It was confirmed that the root growth and MI of different mass of bulb from different garlic had almost no significant differences in a preliminary study.

Test type:

seed germination/root elongation toxicity test

Study type:

laboratory study

Substrate type:

not specified

Limit test:

no

Total exposure duration:

24 h

Test temperature:

22 ± 1C

Details on test conditions:

The bulbs of garlic were allowed to germinate in DI-water until the radicals had elongated to 2.0 cm in length. The seedlings were selected for uniformity in size and shape. Twenty-five seedlings (five seedlings per treatment) were directly placed in ZnO NPs suspensions (10, 20, 30, 40, 50 mg/L) or Zn2+ reference solutions (0, 0.5, 1, 1.5, 2 mg/L) for 24 h. At the end of each treatment, the seedlings were washed carefully with DI-water, and transferred to beakers with DI-water and allowed to germinate at room temperature of 22 ± 1C. Controlled seedlings were placed in DI-water. The roots were protected from direct sunlight in order to minimize fluctuation of the rate of cell division. After 24 h, the root length of the treated seedlings and the controls were measured.

Nominal and measured concentrations:

ZnO NPs suspensions (10, 20, 30, 40, 50 mg/L)
Zn2+ reference solutions (0, 0.5, 1, 1.5, 2 mg/L)

Reference substance (positive control):

no

Species:

other: Allium sativum

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

15 other: mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: root elongation

Species:

other: Allium sativum

Duration:

24 h

Dose descriptor:

LOEC

Effect conc.:

10 other: mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: root elongation

Details on results:

Before the ZnO NPs treatment, the average root length was about 2.1 ± 0.4 cm (mean ± SE), after the treatment, the control seedling root length was 4.3 ± 0.1 cm, while the root lengths were 3.3 ± 0.1 cm, 2.9 ± 0.1 cm, 2.6 ± 0.1 cm, 2.5 ± 0.1 cm, 2.2 ± 0.1 cm at ZnO NPs concentrations of 10, 20, 30, 40, 50 mg/L, respectively.

Reported statistics and error estimates:

The statistical analysis was performed using the procedure of Analysis of Variance (ANOVA) with Microsoft Excel. Data were expressed as mean ± standard error (SE). The statistical significance of the differences among values (mitotic index, and total abnormalities) in the treated samples and the control was evaluated by means of the t-test. Statistical significance was assumed at P < 0.05.

Validity criteria fulfilled:

yes

Conclusions:

Good quality study, however no guideline was followed

Executive summary:

Allium sativum was exposed for 24hr to nZnO quantum dot concentrations up to 50 mg/L. An EC50 was estimated at 15 mg/L with root elongation as endpoint.

Reference 5

Endpoint:

toxicity to terrestrial plants: long-term

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:

according to guideline

Guideline:

EPA OPPTS 850.4200 (Seed Germination/Root Elongation Toxicity Test)

GLP compliance:

not specified

Remarks:

GLP compliance not specified in publication

Specific details on test material used for the study:

nZnO was procured as an aqueous suspension from Meliorum Technologies, Inc., NY, USA (purity — 99.9%) and was used as-obtained. The ionic salts were purchased from Fisher Scientific, Inc., USA (ZnSO4, ACS grade, purity: 99–102%). pH :7.03
Hydrodymic diameter (nm): 11.0 ± 0.7
TEM diameter : 17.4 ± 4.9
average zeta potential (mV): -13.21
Plasmon resonance spectra: 340 nm 3.64 au (absorbance)
ZnO NPs has a HDD (11 ± 0.7 nm) and particles appearing roughly oval for nZnO.
Evaluation of HDD and ζ potential values revealed that both types of NPs were fairly stable inMHW.

Analytical monitoring:

not specified

Vehicle:

no

Species:

Brassica oleracea var. capitata

Plant group:

Dicotyledonae (dicots)

Details on test organisms:

Untreated seeds of maize and cabbage were purchased from Eden Brothers, Dahlonega, GA, USA.

Species:

Zea mays

Plant group:

Monocotyledonae (monocots)

Details on test organisms:

Untreated seeds of maize and cabbage were purchased from Eden Brothers, Dahlonega, GA, USA.

Test type:

seed germination/root elongation toxicity test

Study type:

laboratory study

Substrate type:

filter paper

Limit test:

no

Total exposure duration:

7 d

Test temperature:

23°C

pH:

7.03

Species:

Brassica oleracea var. capitata

Duration:

6 d

Dose descriptor:

EC50

Effect conc.:

136 other: µg/ml

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

nano ZnO

Basis for effect:

germination

Remarks:

rate

Species:

Brassica oleracea var. capitata

Duration:

6 d

Dose descriptor:

EC50

Effect conc.:

9.72 other: µg/ml

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

ZnSO4

Basis for effect:

germination

Remarks:

rate

Details on results:

EC50 for nZnO could not be determined for Zea mays for germination rate
EC50 for nZnO could not be determined for Zea mays for root elongation

Reported statistics and error estimates:

Our data were normally distributed (Kolmogorov–Smirnov (K–S) test: p > 0.1 in all cases); except for a measure of root elongation in cabbage with nZnO treatment (K–S test: Z = 1.644, p b 0.01), which were then log-transformed for use in further analyses as they satisfied normality (K–S test: Z = 1.194, p > 0.1; Pokhrel et al., 2013). Data were analyzed for significant differences in the variances and means between the treatment and the control. When sample variances were significantly different from the control, the t- and p-values were adjusted for independent samples t-test, which tested the null hypothesis that the treatment means are not significantly different from the control at the p ≤ 0.05 level. The Chi-square goodness-of-fit test was used to test if all treatments, including the control, contained the same proportion of the metaxylem counts

In maize, the primary root cells were structurally modified in shape and/or size at the zone of elongation upon exposure tometal NPs or the dissolved ions evaluated. Cells were elongated with NP treatments; while with ZnSO4 treatment cells exhibited

shorter and widermorphology, compared to the control. These contrasting observations for metal NPs versus corresponding

ionic salts suggested that the same mechanism(s) could not be responsible for the observed toxicity pattern, and/or this effectmight be due to differential metal uptake as observed in maize seedlings. Phenotypic root elongation measurement was in good agreement with

the microscopic observations of the cells at the zone of elongation. In maize, unlike with ZnSO4 treatment that resulted in significant dose-dependent effects, nZnO treatment had little to no effect on root growth.

In cabbage, root elongation was not affected by nZnO or ZnSO4 in an excess of 100 µg/ml exposure level. Root development was completely halted as seeds were unable to germinate at higher ZnSO4 concentrations unlike nZnO treatment showed only 40.6% growth inhibition at the highest concentraiton testede. These data revealed lower toxicity of ZnO NP than their corresponding free ions on the primary root growth and development.

In maize, exposure to a wider range of nZnO concentrations (0.01–1000 μg/mL) did not inhibit seed germination (p > 0.1 in all cases), unlike in cabbage a dosedependent germination inhibition occurred. These data showing little to no inhibition of germination with nZnO treatment in maize. In contrast, exposure to ZnSO4 caused significant germination inhibition in both maize (p <0.05) and cabbage (p< 0.05). While complete germination inhibition occurred at 500 μg ZnSO4/mL in cabbage, it only led to ca. 50% germination inhibition in maize at the same concentration.

Validity criteria fulfilled:

yes

Conclusions:

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

Executive summary:

In summary, these results show that NPs of ZnO are potentially toxic to the early development and growth in maize and cabbage, albeit their toxic responses were generally lower for NPs than their specified ionic salts

Reference 6

Endpoint:

toxicity to terrestrial plants: short-term

Type of information:

experimental study

Adequacy of study:

disregarded due to major methodological deficiencies

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: Good quality study, comparison to ZnCl2 only, no bulk

Qualifier:

according to guideline

Guideline:

other: OECD (2003) Guideline for the testing of chemicals, proposal for updating guideline 208, Terrestrial Plant Test, Seedling Emergence and Seedling Growth test

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.4200 (Seed Germination/Root Elongation Toxicity Test)

GLP compliance:

not specified

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.

Species:

Lepidum sativum

Plant group:

Dicotyledonae (dicots)

Test type:

seedling emergence toxicity test

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

72 h

Test temperature:

25+-2°C

Reference substance (positive control):

no

Species:

Lepidum sativum

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

230 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

element

Remarks:

Zn

Basis for effect:

other: root elongation

Reported statistics and error estimates:

All toxicity test results are expressed as mean ± standard error (SE). The toxicity data obtained for both ZnO NP and ZnCl2 treatments were analyzed with Student’s t-test using the software SigmaStat v. 3.5. Levels of p<0.05 and p< 0.01 were considered statistically significant and highly significant, respectively.

The BET characterization of the spiked soil samples indicate that the ZnO NPs dispersed in the soil are still in their pristine state and no aggregation has occurred. Data in Table below show that the addition of ZnO NPs to standard soil gives rise to a linear increase in the specific surface area with the NPs weight fraction for the contaminated soil, while the calculated BET radius of the NP itself remains unchanged.

Table: Weight ratio of NPs in soil dispersions X and the specific surface area of soil samples measured via the BET method

X (%)	SSd (m³/g)
0	0.78
0.1	1.19
0.2	2.62
0.2	2.49a
1	9.9

The adopted spiking procedure seems therefore to preserve the NP size. The dispersion state of the NPs remains unchanged after the wetting procedure of the soil itself, carried out in the toxicity tests, as could be observed by measuring the SSd in the wetted soil. SEM analysis of the contaminated soil sample (i.e., 230 mgZn kgsoil −1, corresponding to 0.03 wt.%) is not very informative. In fact, due to the very low concentration of nanoparticles, even after a very careful examination of this soil sample, it was not possible to identify any particle, agglomerate or mass portion that could be ascribed to the ZnO. It is interesting to note that a bimodal distribution of particles is observed only in the aqueous extract of a 10 wt.% of ZnO contaminated soil, for which the DLS analysis show well resolved peaks at 470 and 103 nm. Other test soils (i.e., 0 and 0.03 wt.%) have shown monomodal distributions. Therefore, the peak at 103 nm can be ascribed to the ZnO NPs, whereas the other peak is assigned, by comparison, to the soil component.

L. sativum seeds showed a 100% germination (no effect with respect to control) with both the soil contaminants. A clear difference between ZnO NPs and soluble Zn can be observed instead for root elongation; in particular, ZnO NPs spiked soil exerted a moderate toxic effect, while ZnCl2 spiked soil produced a 35% biostimulation.

Validity criteria fulfilled:

yes

Conclusions:

Good quality study but no NOEC could be established

Executive summary:

A control and 230 mg Zn/kg was tested using nZnO as test substance. A slight effect on root elongation was noted; whereas a biostimulation effect was seen if ZnCl2 was used as test substance. No germination effects were seen at this concentration.

Reference 7

Endpoint:

toxicity to terrestrial plants: long-term

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Qualifier:

according to guideline

Guideline:

other: USEPA (1996)

GLP compliance:

not specified

Remarks:

GLP compliance not specified in publication

Analytical monitoring:

yes

Vehicle:

no

Species:

Glycine max (G. soja)

Plant group:

Dicotyledonae (dicots)

Details on test organisms:

Soybean (Glycine max) seeds were purchased from Howe Seeds (Mclaughlin, SD). Seeds were treated for 30 min with a 4% NaClO solution for disinfection, followed by rinsing with sterilized MPW. Germination paper (Nasco, Fort Atkinson, WI) cut to fit regular Petri dishes was used as inert material.

Test type:

seed germination/root elongation toxicity test

Study type:

laboratory study

Substrate type:

filter paper

Limit test:

no

Total exposure duration:

5 d

Test temperature:

25°C

pH:

6.5-7.0

Nominal and measured concentrations:

0 (control), 500, 1000, 2000, and 4000 mg L−1

Reference substance (positive control):

no

Species:

Glycine max (G. soja)

Duration:

5 d

Dose descriptor:

NOEC

Effect conc.:

> 4 000 other: mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

nZnO

Basis for effect:

germination

Species:

Glycine max (G. soja)

Duration:

5 d

Dose descriptor:

LOEC

Effect conc.:

2 000 other: mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

nZnO

Basis for effect:

other: root elongation

Species:

Glycine max (G. soja)

Duration:

5 d

Dose descriptor:

NOEC

Effect conc.:

1 000 other: mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

nZnO

Basis for effect:

other: root elongation

Details on results:

An inverse U-shape response was observed in ZnO NP treated seedlings, with maximum size at 500 mg L−1 (30% over control) and a minimum at 4000 mg L−1 (40% shorter than control), (p ≤ 0.05).

Reported statistics and error estimates:

Treatments were arranged in a completely random design. Data were reported as mean ± standard error (SE) and SPSS 15.0 package was used to perform the one-way ANOVA and Tukey’s H.S.D. test.

In the present research, soybean seeds were treated either with hexagonal ZnO NPs (8 nm) at 0, 500, 1000, 2000, and 4000 mg L−1. ICP analyses indicated a purity of 100.0 % ± 3.0 for ZnO NPs. On the other hand the indexing of the XRD pattern from the ZnO nanoparticle ensemble (solid line) to the tetragonal ZnO phase - lattice parameter (a=3.249Å, c=4.206Å) and spacegroup (P63mc) – leaves at least two Bragg peaks unindexed. This indicates that an impurity phase is present in the sample.

Validity criteria fulfilled:

no

Conclusions:

impurity in test substance

Reference 8

Endpoint:

toxicity to terrestrial plants: short-term

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: publication

Qualifier:

no guideline followed

Principles of method if other than guideline:

Cell internalization and upward translocation of ZnO nanoparticles by Lolium perenne were examined. The dissolution of ZnO nanoparticles and its contribution to the toxicity on ryegrass were also investigated. Zn2+ ions were used to compare and verify the root uptake and phytotoxicity of ZnO nanoparticles in a hydroponic culture system. The root uptake and phytotoxicity were visualised by light, scanning electron, and transmission electron microscopies.

GLP compliance:

not specified

Specific details on test material used for the study:

nanoscale metal Zn was from Zunye Nanomaterials Co. Ltd., Shenzhen, China, and the nanoscale metal oxide ZnO was from Hongchen Material Sci. & Tech. Co. Ltd., Zhejiang, China.

Details on preparation and application of test substrate:

Compositions of the nutrient solution (1 strength Hoagland solution): 20 ppm (NH4)2SO4, 10 ppm NH4NO3, 3.1 ppm NaH2PO4, 40 ppm K2SO4, 15 ppm CaCl2.2H2O, 0.35 ppm EDTA.FeNa.3H2O, 25 ppm MgSO4.3H2O, 20 ppm Al2(SO4)3.18H2O, 0.1 ppm ZnSO4.7H2O, 0.1 ppm H3BO3, 0.025 ppm CuSO4.5H2O, 1 ppm MnSO4.H2O and 0.05 ppm Na2MoO4.2H2O. All of these compositions were analytical grade with a purity of > 98%. The pH value of the nutrient solution was adjusted to near neutral.

Species:

Lolium perenne

Plant group:

Monocotyledonae (monocots)

Details on test organisms:

- Common name: ryegrass
- Plant family: Poaceae

Test type:

other: root uptake of ZnO

Study type:

laboratory study

Substrate type:

other: nutrient solution

Limit test:

no

Total exposure duration:

12 d

Test temperature:

25-30°C in daytime (16 h), 15-20°C in nighttime (8 h)

pH:

7

Details on test conditions:

TEST SYSTEM
- Test container (type, material, size): 1000 mL beakers
- No. of seeds per container: 9
- No. of replicates per treatment group: 3

NUTRIENT MEDIUM (if used)
- Description: 20 ppm (NH4)2SO4, 10 ppm NH4NO3, 3.1 ppm NaH2PO4, 40 ppm K2SO4, 15 ppm CaCl2.2H2O, 0.35 ppm EDTA.FeNa.3H2O, 25 ppm MgSO4.3H2O, 20 ppm Al2(SO4)3.18H2O, 0.1 ppm ZnSO4.7H2O, 0.1 ppm H3BO3, 0.025 ppm CuSO4.5H2O, 1 ppm MnSO4.H2O and 0.05 ppm Na2MoO4.2H2O.


GROWTH CONDITIONS
- Photoperiod: 16 h light / 8 h dark
- Day/night temperatures: 25-30°C day / 15-20°C night

Nominal and measured concentrations:

10, 20, 50, 100, 200, 1000 mg/L (nominal), control

Species:

Lolium perenne

Duration:

12 d

Dose descriptor:

other: IC50

Effect conc.:

51 other: mg/L

Nominal / measured:

nominal

Conc. based on:

element (dissolved fraction)

Remarks:

from nano ZnO

Basis for effect:

growth

Species:

Lolium perenne

Duration:

12 d

Dose descriptor:

other: IC50

Effect conc.:

38 other: mg/L

Nominal / measured:

nominal

Conc. based on:

element (dissolved fraction)

Remarks:

from ZnSO4

Basis for effect:

growth

The toxicity of Zn0 nanoparticles and Zn2+ ions to the ryegrass seedlings were evident and increased with increasing concentration of both ZnO nanoparticles and Zn2+, which could be easily observed by visual examination. The growth of seedlings in both treatments, especially with concentrations higher than 50 mg/L, was retarded with shorter roots and shoots than the control. Toxic symptoms seem more severe with Zn2+ than ZnO nanoparticles, in that shoots became yellow with concentrations of Zn2+ higher than 50 mg/L and almost withered to death at a concentration up to 1000 mg/L. As regards the dose-response curves of ZnO nanoparticles and Zn2+, there appeared a concentration threshold of both treatments, below which no significant toxic symptoms were observed. However, the seedling biomass decreased with increasing dose after the threshold. The threshold of Zn2+ for both shoot and root was ca. 20 mg/L, whereas it was around 10 and 50 mg/L of ZnO nanoparticles for ryegrass shoots and roots, respectively. The IC100 for ZnO and Zn2+ is arbitrarily taken to be near 200 mg/L, after which biomass kept nearly unchanged with further increasing concentrations. The 50% biomass inhibitory concentration (IC50) was defined in as the concentration at which the biomass equals the mean biomasses of blank and at IC100. IC50 of Zn2+ was estimated to be ca. 38 mg/L, which was lower than that of ZnO nanoparticles (ca. 64 ZnO mg/L, 51 mg Zn/L). Toxic symptoms were further examined by LM of longitudinally sectioned primary root tips. Controls showed normal development with dividing cells. However, shrank morphology of the root tips indicates severe impact of ZnO nanoparticles and Zn2+ ions. In the presence of 1000 mg/L ZnO nanoparticles or Zn2+, the epidermis and root cab were broken, the cortical cells were highly vacuolated and collapsed, and the vascular cylinder also shrank. No living cells in the root tips could be observed in the presence of 1000 mg/L Zn2+, whereas part of the vascular cells seems still alive with 1000 mg/L ZnO nanoparticles, though not active as the control. Both treatments increased total Zn in ryegrass tissues, but with different trends. There was no significant difference of root Zn contents between the two treatments with concentrations lower than 100 mg/L. When the concentrations of ZnO nanoparticles and Zn2+ ions in the nutrient solution were higher than 100 mg/L, root Zn content reduced with increasing Zn2+ concentration, however, increased with increasing ZnO concentration. Root Zn content in the presence of 1000 mg/L ZnO nanoparticles was 3.6 times higher than that of the 1000 mg/L Zn2+. Shoot Zn contents remained low under the ZnO nanoparticle treatments (0.25-1.36 mg/kg), and were much lower than that under die Zn2+ treatments (0.25-19.1 mg/kg). Translocation factor (TF) of Zn, defined as Zn content ratio of shoot to root, were very low (0.02-0.01) under the ZnO nanoparticle treatments, showing a decreasing tendency with increasing concentration of ZnO. However, under the Zn2+ treatments, TF (0.03-0.50) increased with increasing concentration of Zn2+. A much (1.4-50 times) lower Zn TF under the ZnO treatments than Zn2+ treatments indicates that the increasing root Zn under the ZnO treatments could be mainly from the increasing adsorption and uptake of ZnO nanoparticles by the ryegrass roots and little ZnO nanoparticles could (if any) be transported to the shoots. SEM images showed that root surface in the control and Zn2+ treatments were free of particle adherence. However, adsorption of ZnO nanoparticles and their aggregations on the root surface was evident and the coverage increased with increasing ZnO dose. The particles were observed filled in the epidermal crypt or adhered onto the surface. TEM images of the cross sections of the ryegrass roots show the presence of dark dots (particles) in the endodermis and vascular cylinder under the ZnO treatments. The dark dots distributed in the apoplast, cytoplasm, and even nuclei. One or several nanoparticles could be identified in the dark dots covered by cytoplast as shown by higher magnification TEM image. The size of these nanoparticles were measured to be 19 ± 6 nm (n = 89), identical to the size of ZnO nanoparticles. Such dark dots (i.e., particles) were not observed under either the control or Zn2+ treatments. Thus, it was concluded that the ZnO nanoparticles could enter into the endodermis and vascular cylinder of the ryegrass roots. In summary the authors concluded, ZnO nanoparticles were found able to concentrate in the rhizosphere, enter the root cells, and inhibit seedling growth; the phytotoxicity of ZnO nanoparticles could not primarily come from their dissolution in the bulk nutrient solution or the rhizosphere.

Validity criteria fulfilled:

yes

Conclusions:

Study considered useful for assessing terrestrial toxicity.

Executive summary:

Toxic symptoms seem more severe by Zn2+ than by ZnO NP.

the ZnO nanoparticles could enter into the endodermis and vascular cylinder of the ryegrass roots. In summary the authors concluded, ZnO nanoparticles were found able to concentrate in the rhizosphere, enter the root cells, and inhibit seedling growth; the phytotoxicity of ZnO nanoparticles could not primarily come from their dissolution in the bulk nutrient solution or the rhizosphere.

Reference 9

Endpoint:

toxicity to terrestrial plants: 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: extended abstract

Qualifier:

no guideline followed

Principles of method if other than guideline:

Seeds of Lolium perenne were germinated for about two weeks after sterilisation and soaking. Seedlings were then selected and transplanted to 1000 mL beakers containing nutrient solutions and different concentrations of nano-ZnO or Zn2+. Biomass of shoots and roots were measured as well as the respective Zn-concentrations.

GLP compliance:

no

Specific details on test material used for the study:

nanoscale metal Zn was from Zunye Nanomaterials Co. Ltd., Shenzhen, China, and the nanoscale metal oxide ZnO was from Hongchen Material Sci. & Tech. Co. Ltd., Zhejiang, China.

Details on preparation and application of test substrate:

Compositions of the nutrient solution were as follows: 20 ppm (NH4)2SO4, 10 ppm NH4NO3, 3.1 ppm NaH2PO4, 40 ppm K2SO4, 15 ppm CaCl2.2H2O, 0.35 ppm EDTA.FeNa.3H2O, 25 ppm MgSO4.3H2O, 20 ppm Al2(SO4)3.18H2O, 0.1 ppm ZnSO4.7H2O, 0.1 ppm H3BO3, 0.025 ppm CuSO4.5H2O, 1 ppm MnSO4.H2O and 0.05 ppm Na2MoO4.2H2O. All of these compositions were analytical grade with a purity of > 98%. The pH value of the nutrient solution was
adjusted to 7.

Species:

Lolium perenne

Plant group:

Monocotyledonae (monocots)

Details on test organisms:

- Common name: ryegrass
- Plant family: Poaceae

Test type:

early seedling growth toxicity test

Study type:

laboratory study

Substrate type:

other: nutrient solution

Limit test:

no

Total exposure duration:

12 d

Test temperature:

25-30°C in daytime (16 h), 15-20°C in nighttime (8 h)

pH:

7

Details on test conditions:

TEST SYSTEM
- Testing facility:
- Test container (type, material, size): 1000 mL beakers
- No. of seeds per container: 9
- No. of replicates per treatment group: 3


NUTRIENT MEDIUM (if used)
- Description: 20 ppm (NH4)2SO4, 10 ppm NH4NO3, 3.1 ppm NaH2PO4, 40 ppm K2SO4, 15 ppm CaCl2.2H2O, 0.35 ppm EDTA.FeNa.3H2O, 25 ppm MgSO4.3H2O, 20 ppm Al2(SO4)3.18H2O, 0.1 ppm ZnSO4.7H2O, 0.1 ppm H3BO3, 0.025 ppm CuSO4.5H2O, 1 ppm MnSO4.H2O and 0.05 ppm Na2MoO4.2H2O.


GROWTH CONDITIONS
- Photoperiod: 16 h light / 8 h dark
- Day/night temperatures: 25-30°C / 15-20°C

Nominal and measured concentrations:

10, 20, 50, 100, 200 and 1000 mg/L (nominal), control

The growth of seedlings was greatly inhibited under the nano-ZnO treatments. Ryegrass shoot biomass significantly (p 0.05) reduced with increasing concentration of nano-ZnO in the nutrient solution, especially at the nano-ZnO concentrations higher than 20 mg/L. The shoot growth inhibition by the nano-ZnO seems the worst at 200 mg/L, with nearly constant biomass at concentrations up to 1,000 mg/L. Biomass reduction of ryegrass root showed a similar tendency. But significant root inhibition was observed only at the nano-ZnO concentrations of 100-1,000 mg/L and the root biomass kept nearly unchanged in this concentration range. No significant difference of the ryegrass biomass between the treatments of Zn2+ and nano-ZnO could be observed. But the yellow and withered shoots at higher Zn2+ concentrations indicates that Zn2+ might be more toxic to the ryegrass than nano-ZnO. Hence, one may wonder if the phytotoxicity of nano-ZnO came from the dissolution of nano-ZnO. Zinc ion concentrations in the nano-ZnO treated nutrient solutions after centrifugation and filtration were lower than 6 mg/L. No significant toxicity of Zn2+ at 6 mg/L was observed to the ryegrass. Thus, the phytotoxicity of nano-ZnO could not result from its direct dissolution in the nutrient solution whose pH was adjusted to be 7. Both treatments increased total Zn in the ryegrass tissues, but with different trends. There was no significant difference of root Zn contents between the treatments of nano-ZnO and Zn2+ with concentrations lower than 100 mg/L. When the concentrations of nano-ZnO or Zn2+ in the nutrient solution were higher than 100 mg/L, root Zn content reduced with increasing Zn2+ concentration; however, increased within experimental concentrations of nano-ZnO. The reduction of root Zn content at higher Zn2+ concentrations may be due to the great inhibition of plant activity while the increasing root Zn under the nano-ZnO treatments could be from increasing adsorption of nano-ZnO by ryegrass root. It was evident that the nano-ZnO coverage on rootsurface increased with increasing nano-ZnO concentration in the nutrient solution. Shoot Zn contents (0.25-1.36 mg/kg) remained low under the nano-ZnO treatments and were much lower than that under the Zn2+ treatments (0.25-19.1 mg/kg). This indicates that ZnO particles might not directly transport from root to shoot, or dissolve to be Zn2+, at least not much, at root surface or inside root tissue. Therefore it was concluded, the phytotoxicity of nano-ZnO could not mainly come from its dissolution at root surface or inside root tissue.

Validity criteria fulfilled:

not specified

Reference 10

Endpoint:

toxicity to terrestrial plants: short-term

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:

no guideline followed

Principles of method if other than guideline:

Effects of five types of nanoparticles including ZnO (other: multi-walled carbon nanotube, aluminum, alumina, zinc) on seed germination and root growth of six higher plant species (radish, rape, ryegrass, lettuce, corn, and cucumber) were investigated after treatment with suspensions of nanoparticles.

GLP compliance:

not specified

Specific details on test material used for the study:

nanoscale metal Zn was from Zunye Nanomaterials Co. Ltd., Shenzhen, China, and the nanoscale metal oxide ZnO was from Hongchen Material Sci. & Tech. Co. Ltd., Zhejiang, China.

Details on preparation and application of test substrate:

- Method of application to filter paper (if used): 5 ml of test medium added to filter paper in Petri dish (100 mm x 15 mm)

Species:

Brassica napus

Plant group:

Dicotyledonae (dicots)

Details on test organisms:

- Common name: rape
- Plant family: Brassicaceae
- Source of seed: Chas. C. Hart Seed Co., USA

Species:

Raphanus sativus

Plant group:

Dicotyledonae (dicots)

Details on test organisms:

- Common name: radish
- Plant family: Brassicaceae
- Source of seed: Chas. C. Hart Seed Co., USA

Species:

Lolium perenne

Plant group:

Monocotyledonae (monocots)

Details on test organisms:

- Common name: ryegrass
- Plant family: Poaceae
- Source of seed: Chas. C. Hart Seed Co., USA

Species:

Lactuca sativa

Plant group:

Dicotyledonae (dicots)

Details on test organisms:

- Common name: lettuce
- Plant family: Asteraceae
- Source of seed: Chas. C. Hart Seed Co., USA

Species:

Zea mays

Plant group:

Monocotyledonae (monocots)

Details on test organisms:

- Common name: corn
- Plant family: Poaceae
- Source of seed: Chas. C. Hart Seed Co., USA

Species:

Cucumis sativus

Plant group:

Dicotyledonae (dicots)

Details on test organisms:

- Common name: cucumber
- Plant family: Cucurbitaceae
- Source of seed: Chas. C. Hart Seed Co., USA

Test type:

seed germination/root elongation toxicity test

Study type:

laboratory study

Substrate type:

filter paper

Limit test:

no

Total exposure duration:

5 d

Test temperature:

room temperature

pH:

6.5 - 7.5

Details on test conditions:

TEST SYSTEM
- Test container (type, material, size): Petri dish (100 mm x 15 mm)
- No. of seeds per container: 10 seeds per dish with 1 cm distance
- No. of replicates per treatment group: 3

GROWTH CONDITIONS
- Photoperiod: dark

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
seed germination and root growth after 5 days

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Test concentrations: 20, 200, 2000 mg/L

Nominal and measured concentrations:

control, 20, 200, 2000 mg/L (nominal)

Species:

Lactuca sativa

Duration:

5 d

Dose descriptor:

NOEC

Effect conc.:

> 2 000 other: mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

germination

Species:

Raphanus sativus

Duration:

5 d

Dose descriptor:

NOEC

Effect conc.:

> 2 000 other: mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

germination

Species:

Brassica rapa

Duration:

5 d

Dose descriptor:

NOEC

Effect conc.:

> 2 000 other: mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

germination

Species:

Cucumis sativus

Duration:

5 d

Dose descriptor:

NOEC

Effect conc.:

> 2 000 other: mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

germination

Species:

Zea mays

Duration:

5 d

Dose descriptor:

LOEC

Effect conc.:

2 000 other: mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

germination

Species:

Lolium perenne

Duration:

5 d

Dose descriptor:

LOEC

Effect conc.:

2 000 other: mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

germination

Species:

Brassica napus

Duration:

5 d

Dose descriptor:

other: IC50

Effect conc.:

ca. 20 other: mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: root growth

Species:

Raphanus sativus

Duration:

5 d

Dose descriptor:

other: IC50

Effect conc.:

ca. 50 other: mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: root growth

Species:

Lolium perenne

Duration:

5 d

Dose descriptor:

other: IC50

Effect conc.:

ca. 20 other: mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: root growth

Species:

Lactuca sativa

Duration:

5 d

Dose descriptor:

other: IC100

Effect conc.:

ca. 2 000 other: mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: root growth

Species:

Zea mays

Duration:

5 d

Dose descriptor:

other: IC100

Effect conc.:

ca. 2 000 other: mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: root growth

Species:

Cucumis sativus

Duration:

5 d

Dose descriptor:

other: IC100

Effect conc.:

ca. 2 000 other: mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: root growth

Seed germinations were not affected by the nanoparticles except for the seeds of ryegrass and corn. Seed germination of ryegrass and corn was inhibited by nano-Zn and nano-ZnO, respectively. The influence of nanoparticle suspensions at 2000 mg/L on root growth varied with types of nanoparticles and plant species. Phytotoxicity of nano-Zn and nano-ZnO was evident. Their suspensions significantly inhibited root growth of corn and practically terminated root development of the other five plant species whose root length at the end of experiment was unable to be accurately measured with a ruler. However, they all germinated with cotyledons sprouting out of seed coat. In this work, 1 mm was used as the minimum length to be called roots. Radish, rape and ryegrass were selected as test plant species in the following experiments.

To examine which process (seed soaking or incubation after the soaking) primarily retarded the root growth, three treatments were used: (1) both seed soaking and incubation were performed in nanoparticle suspensions; (2) seeds were soaked in nanoparticle suspensions for 2 h, and were then transferred into Petri dishes with 5 mL DI-water for incubation after being rinsed three times with DI-water; and (3) seeds were incubated in Petri dishes with 5 mL nanoparticle suspensions after being soaked in DI-water for 2 h. As described, the root growth was almost halted by seed soaking and incubation in the suspensions of nano-Zn and nano-ZnO (the first treatment). Also, root growth of radish, rape and ryegrass was nearly terminated under the third treatment (soaking in water, then incubation in suspension), while roots could grow relatively well under the second treatment (soaking in suspension, then incubation in water) though the root development of the three plant species was significantly inhibited by nano-Zn, and, that of ryegrass by nano-ZnO.

To further clarify the phytotoxicity of nano-Zn and nano-ZnO, experiments were carried out to determine the dose-response relationship of nano-Zn and nano-ZnO using the first treatment: both seed soaking and incubation in the nanoparticle suspensions or Zn2+ solutions. No significant root growth inhibition was observed under low concentrations (less than 10 mg/L for rape and ryegrass and 20 mg/L for radish). Root growth was clearly restricted with increasing concentration, and was almost terminated at 200 mg/L. Nano-Zn and nano-ZnO showed similar phytotoxicity at each of concentrations. Fifty percent root growth inhibitory concentrations (IC50) of both nano-Zn and nano-ZnO were estimated to be near 50 mg/L for radish, and near 20 mg/L for rape and ryegrass.

Two experiments were conducted to exclude Zn2+ from the phytotoxicity of nano-Zn and nano-ZnO. First, the phytotoxicity of supernatants of the nanoparticle suspensions after centrifuging at 3000 g for 1 h and filtering through 0.7 µm glass filters was measured, then the phytotoxicity of Zn2+ solutions by dissolving ZnSO4•7H2O in DI-water was tested. No statistically significant effect (negative or positive) was observed except for the growth enhancement by the supernatant of ZnO suspension at 2000 mg/L on rape ( p = 0.050) and radish ( p = 0.014). Concentrations of Zn2+ in the supernatants (after centrifugation and filtration) were 0.3-3.6 mg/L. Therefore, five concentration points of 0, 1, 2, 3 and 4 Zn2+ mg/L were made from ZnSO4•7H2O to investigate the phytotoxicity of Zn2+. No significant effect on the root of radish, rape and ryegrass from these Zn2+ concentrations was observed.

Validity criteria fulfilled:

yes

Conclusions:

Good quality study useful for assessing terrestrial toxicity

Executive summary:

Zn and ZnO particles were observed to have significant inhibition on seed germination and root growth of the six plant species. The inhibition occurred during the seed incubation process rather than seed soaking stage with IC50 of about 50 mg/L for radish, and near 20 mg/L for rape and ryegrass. The supernatants of the substrates after centrifugation and filtration and zinc ion (Zn2+) in the similar concentration range to that of the supernatants showed no significant phytotoxicity. Thus, it could be concluded that the phytotoxicity

of Zn and ZnO particles was not directly from their dissolution in bulk aqueous solutions though dissolution right on the root surface cannot be ruled out at this point, which needs further investigation.

Reference 11

Endpoint:

toxicity to terrestrial plants: 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:

no guideline followed

Principles of method if other than guideline:

Plants of F. esculentum were transferred to three 50 mL Falcon tubes with four seedlings in 40 mL of 1/23 strength Hoagland solution for hydroponic culture. The hydroponic seedlings in Hoagland solution were reated with nano-and micro-sized ZnO particles at concentrations of 0-2000 mg/L

GLP compliance:

not specified

Remarks:

GLP compliance not specified in publication

Specific details on test material used for the study:

The ZnO NPs and microparticles (MPs) were purchased from Sigma Aldrich Chemical Co. (St. Louis, MO, USA) and stored according to the vendor's instructions. Nanoparticles size and morphology were characterized by transmission electron microscopy (TEM; LIBRA 120, Carl Zeiss, Germany) in 1/2 strength Hoagland solution at concentration 500 mg/L. In order to provide more reliable data on phytotoxicity of NPs, a morphology characterization
of the NPs in solution (colloidal stability, surface potential, and diameter) is required (Sayes and Warheit 2009). In nutrition solution state, individual of ZnO NPs are rod and nonspherical in shape, and ZnO MPs are also nonspherical particles with little extent of aggregation than that of NPs.
The particle size of ZnO NPs was 44.46±4.84 nm and ZnO MPs's particle size was 2~5 μm, which were almost the same as that (ZnO NPs, <50 nm; ZnO MPs, <5 μm) from producer. ZnO NPs purity was >97 % and the specific surface area was >10.8 m2 g−1. ZnO MPs had a purity of 99 % and a specific surface area of 3–4 m2 g−1.

Analytical monitoring:

yes

Details on sampling:

ZnO particles were prepared in 1/2 strength Hoagland solution (Fossati et al. 1980) at treatment concentrations of 0, 1, 5, 100, 1,000, and 2,000 mg/L separately to disperse the particles for hydroponic culture. For protection of aggregation of ZnO NP, suspensions were homogenized by ultrasonication (100 W, 40 kHz) for 30 min and then filtered with 0.45 μm nylon membrane filter (Whatman International, Maidstone, Kent, UK).

Vehicle:

no

Species:

other: Fagopyrum esculentum

Plant group:

Dicotyledonae (dicots)

Details on test organisms:

This species was selected as a model plant system due to its wide distribution and common use in phytotoxicity studies (EPA 1996). Seeds of F. esculentum were purchased from local stores and sown in a pot containing natural soil in a greenhouse maintained at 25 °C for 2 weeks. The plants were transferred to three 50 mL Falcon tubes with four seedlings in 40 mL of 1/2 strength Hoagland solution for hydroponic culture. The hydroponic seedlings in Hoagland solution were treated with nano- and micro-sized ZnO particles at concentrations of 0–2,000 mg/L

Test type:

other: Biomass

Study type:

laboratory study

Substrate type:

other: Hoagland solution

Limit test:

no

Total exposure duration:

5 d

Reference substance (positive control):

no

Species:

other: Fagopyrum esculentum

Duration:

5 d

Dose descriptor:

NOEC

Effect conc.:

5 other: mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: biomass

Species:

other: Fagopyrum esculentum

Duration:

5 d

Dose descriptor:

NOEC

Effect conc.:

100 other: mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: root elongation

Species:

other: Fagopyrum esculentum

Duration:

5 d

Dose descriptor:

LOEC

Effect conc.:

1 000 other: mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: root elongation

Species:

other: Fagopyrum esculentum

Duration:

5 d

Dose descriptor:

LOEC

Effect conc.:

10 other: mg/l

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: biomass

Reported statistics and error estimates:

Each concentration of the three treatments was conducted in triplicate (three tubes with four seedlings each). Turkey's post hoc test and an analysis of one-way ANOVAwere used to test for differences using SPSS version 12.0 software (SPSS Inc., Chicago, IL, USA).

Our results showed that biomass and TF were decrease in response to ZnO high concentration. The seedling growth (biomass) of F. esculentum decreased with an increase in the ZnO NP and MP concentrations. Interestingly, the biomass of F. esculentum treated with 1 mg/L ZnO NPs (2.35 g±0.2) and 1 and 5 mg/L ZnO MPs (2.32 g±0.2) and 2.43 g±0.1, respectively) was rather higher than that of the control (2.08 g±0.2), suggesting that no decrease in biomass occurred at low concentrations due to the essential elemental nutrition in the ZnO particles. The significant biomass reduction in F. esculentum at concentrations of 10– 2,000 mg/L was 7.7–26.4 % for the ZnO NP and 11.4– 23.5 % for the ZnO MP treatment (p<0.05). ZnO NP and MP suspensions released 0.58 to 3.85 μg/mL of concentration Zn2+-free ion in all treatments. In particular, the roots were shortened and damaged in plants exposed to the 1,000 and 2,000 mg/L ZnO NP treatment concentrations.

Validity criteria fulfilled:

yes

Conclusions:

Good quality study

Executive summary:

F. esculentum was exposed upto concentrations of 2000 mg/L. Effects on root elongation were seen at 100 mg nZnO/L.

Reference 12

Endpoint:

toxicity to terrestrial plants: short-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:

no guideline followed

Principles of method if other than guideline:

C. sativus was grown in a pot containing natural soil in a greenhouse maintained at 25°C for 4 weeks. They were transferred into three of 100 ml beaker with nine seedlings each in 50 mL of 1/2 strength Hoagland solution and grown at a controlled temperature of 25°C with aeration. After 5 days they were exposed to 0,10,50,100,500 and 1000 mg/L ZnO NP. Before use, they were ultrasonicated (100 W, 40 kHz) for 30 min. Nano Zn suspensions became homogeneously turbid after sonication.

GLP compliance:

not specified

Remarks:

GLP compliance not specified in publication

Specific details on test material used for the study:

All chemicals and reagents used in this study were purchased from Sigma Aldrich Chemical Co. (St. Louis, MO, USA), stored according to the vendor’s instructions, and used as received. The size of the ZnO NPs was 50 nm. The surface area of the Zn NPs was 35–50 m2/g.

Analytical monitoring:

yes

Details on sampling:

Metal concentrations in the filtered (0.45 μm pore size, Whatman) supernatants of the hydroponic solutions after centrifugation were determined by atomic absorption spectroscopy. The system was calibrated using certified reference materials (NO 10-C: Rice Flour) obtained from the National Institute for Environmental Studies of Japan.
Bulk nutrient solutions in the beakers were collected after setting for 8 h without stirring. The bulk nutrient solution-exposed metal and metal oxide NPs were observed by transmission electron microscopy (TEM) (LIBRA 120, Carl Zeiss, Germany). In addition, metal and metal oxide NPs in deionized water were observed by TEM. The pH levels of the solutions were measured.

Vehicle:

no

Species:

Cucumis sativus

Plant group:

Dicotyledonae (dicots)

Details on test organisms:

Seeds from C. sativus were purchased fromlocal stores

Test type:

seedling emergence toxicity test

Study type:

laboratory study

Substrate type:

other: Hoagland solution

Limit test:

no

Total exposure duration:

5 d

Test temperature:

25°C

Nominal and measured concentrations:

0, 10, 50, 100, 500, and 1,000 mg/L of Zn, and ZnO NPs as well as Zn2+

Reference substance (positive control):

no

Species:

Cucumis sativus

Duration:

5 d

Dose descriptor:

EC50

Effect conc.:

629 other: mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: biomass

Species:

Cucumis sativus

Duration:

5 d

Dose descriptor:

LOEC

Effect conc.:

10 other: mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: biomass

Reported statistics and error estimates:

Each concentration point of the three treatments was conducted in triplicate (three beakers with nine seedlings each). Statistical analysis of the experimental data utilized Student’s t test.

the biomass levels were 56% and 42% of that of control at 1,000 mg/L of Zn NPs and ZnO NPs, respectively. The IC50 of Zn NPs, ZnO NPs, and Zn2+ were 1,700, 629, and 262 mg/L, respectively.

The concentration of Zn in C. sativus increased in response to treatment with Zn2 +. In nanometal and nanometal oxide treatments, the Zn concentration in C. sativus increased gradually with increased metal concentration (up to 100 mg/L), after which accumulated levels reached saturation The concentrations of saturated Zn (after 100 mg/L) were about 5,000 and 4,000 μg/g upon treatment with ZnO NPs and Zn NPs, respectively.

The aggregation of metal oxide NPs was greater than that of metal NPs. Specially, ZnO NPs greatly aggregated as well, although individual particles with a nearly spherical shape were also observed. The size of the aggregated Zn and ZnO NPs was 500 nm.

Validity criteria fulfilled:

yes

Conclusions:

No guideline followed but study considered useful for assessing terrestrial toxicity

Executive summary:

ZnO NPs showed the highest toxicity and increase Zn uptake in C. sativus due to solubility and defense mechanism. We suggested that oxidative stress potential in plants might be a better indicator of phytotoxicity than bioavailability in plants treated with NPs.

Reference 13

Endpoint:

toxicity to terrestrial plants: short-term

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: No guideline was followed. Zn concentrations were not measured.

Qualifier:

no guideline followed

Principles of method if other than guideline:

A. cepa was grown for three days without the NPs. When the length of the roots reached between 1.3 and 1.5 cm, A. cepa was transferred to fresh solutions of zinc NPs. The control was treated only with DI water. The experiments were conducted with three onion bulbs per exposure at 25±2 °C under 12-h light/dark cycles in glass vessels. The phenotype and weight of the total fresh biomass of A. cepa roots growing with and without the NPs were measured at different intervals over a span of three days, and the average values were calculated.

GLP compliance:

not specified

Specific details on test material used for the study:

97% zinc oxide NPs having diameters of approximately 50nm were obtained from Sigma Aldrich.

Analytical monitoring:

no

Vehicle:

no

Species:

Allium cepa

Plant group:

Monocotyledonae (monocots)

Details on test organisms:

Fresh and healthy A. cepa (onion bulbs) having diameters of 20–25mmand weights of 10–15 g were obtained from a local market.
The onions were washed under running tap water and then the dried outer scales were removed before they were used in the experiment.

Test type:

seed germination/root elongation toxicity test

Study type:

laboratory study

Substrate type:

not specified

Limit test:

no

Total exposure duration:

72 h

Test temperature:

25 +-2°C

Nominal and measured concentrations:

5, 10, 20gml−1

Reference substance (positive control):

no

Species:

Allium cepa

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

5 other: µg/ml

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: root elongation

Details on results:

The root lengths were reduced to 1.50, 1.25, and 1.24cm at 5, 10, and 20gml−1, respectively, as compared to the control which was measured at 4.75cm

The representative TEM image of the zinc oxide NPs shows that most of the particles were rod shaped, spherical, or hexagonal

with sizes ranging between approximately 50 and 100 nm, and the particles were found to be clustered. During the exposure

of the A. cepa roots to zinc oxide NPs, the NPs aggregated and precipitated.

The angular positions of the Bragg peaks and the face-centered-cubic structure of the cobalt and zinc nanostructures indicate that the

surfaces of both the NPs were evidently particulate and crystalline in nature.

The phytotoxicity of the oxide NPs was evident and increased with the increasing concentrations. In the presence of the zinc oxide

NPs, the root lengths were reduced to 1.50, 1.25, and 1.24cm at 5, 10, and 20gml−1, respectively, as compared to the control which was measured at 4.75cm. The plant roots exposed to the zinc oxide NPs at 20gml−1 showed almost no growth; even during the initial exposure (24 h).

Validity criteria fulfilled:

yes

Conclusions:

Good quality study, however no guideline followed.

Executive summary:

Allium cepa was exposed to nZnO particles for 72h. Effects on root elongation were seen at 5 µg nZnO/L.

Reference 14

Endpoint:

toxicity to terrestrial plants: long-term

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

no guideline followed

Principles of method if other than guideline:

The objectives of the present study were to (i) compare the toxicity of ZnO-NPs to plants and microorganisms with the effects of bulk ZnO and the soluble salt ZnCl2, which were used as reference compounds for size-dependent and solubility effects; (ii) assess the influence of the chemical form of Zn on its distribution in soil fractions; and (iii) relate the toxic effects to the internal concentrations of Zn in the plants.

GLP compliance:

not specified

Remarks:

GLP compliance not specified in paper

Specific details on test material used for the study:

Uncoated ZnO nanopowder (advertised particle size <100 nm diameter) and the ZnO bulk form were purchased from Sigma-Aldrich (Germany), and the ZnCl2 was from Panreac Spain).

Analytical monitoring:

yes

Vehicle:

no

Species:

Triticum aestivum

Plant group:

Monocotyledonae (monocots)

Details on test organisms:

Common name: wheat
The seeds were obtained from the Spanish National Centre for Seeds and Vegetal Varieties (Madrid).

Species:

Raphanus sativus

Plant group:

Dicotyledonae (dicots)

Details on test organisms:

Common name: radish
The seeds were obtained from the Spanish National Centre for Seeds and Vegetal Varieties (Madrid).

Species:

Vicia sativa

Plant group:

Dicotyledonae (dicots)

Details on test organisms:

Common name: vetch
The seeds were obtained from the Spanish National Centre for Seeds and Vegetal Varieties (Madrid).

Test type:

other: The emergence of the seedswas registered at 7 and 14 days. After 21 days, the emerged plants were harvested, collecting the whole plant. The lengths of stems and roots and the fresh weights of the aerial parts were recorded.

Study type:

laboratory study

Substrate type:

natural soil

Remarks:

Soil was collected from the top layer (0–20 cm depth) of a field located near Madrid (Spain), GPS coordinates N 40°27′18″, W 03°44′55″. The soil was air-dried and sieved (<2 mm mesh).

Limit test:

yes

Total exposure duration:

21 d

Test temperature:

18-22°C

pH:

7.48-8.29

Details on test conditions:

Soil was contaminated with 1000 mg Zn kg−1 soil as ZnONPs, ZnO bulk, or ZnCl2 to test for potential harmful effects on microorganisms and plants. Additionally, a fourth ZnCl2 exposure treatment for plants at 250 mg Zn kg−1 soil (ZnCl2-250) was prepared to determine if the solubility of ZnO-NPs could induce effects that were similar to a lower concentration of the salt after 21 days, assuming that the Zn2+ from the oxide would be responsible for toxicity. The Zn concentrations were always calculated on a dry soil (DW) basis. Untreated soil (natural Zn occurrence) was used as the control. The ZnONP and ZnO bulk powders were directly added to the soil and hand blended following previous studies (Hooper et al. 2011; Garcia-Gomez et al. 2014a). The powder application was selected because it was easier to make a homogenized mixing of the samples. The mixes were later sieved (2 mm) three times to ensure the homogenization of the samples. ZnCl2 was added to soil as an aqueous solution to guarantee that Zn2+ was the chemical form added to the soil. The volume of water added was equivalent to that required to achieve 50 % of the water holding capacity (WHC) of the soil. Control and treatment samples were wet to 50 % WHC and stored in the dark

Nominal and measured concentrations:

1000 mg Zn/kg dw for ZnCl2, inano and bulk (and 250 mg Zn/kg dw for ZnCl2 only)

Reference substance (positive control):

no

Species:

Triticum aestivum

Duration:

21 d

Dose descriptor:

other: % inhibition germination (from control)

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

element (total fraction)

Remarks:

ZnO nano

Basis for effect:

germination

Remarks:

% inhibition

Remarks on result:

other: Not siginificantly different from control

Species:

Triticum aestivum

Duration:

21 d

Dose descriptor:

other: % inhibition germination (from control)

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

element (total fraction)

Remarks:

ZnO bulk

Basis for effect:

germination

Remarks:

% inhibition

Remarks on result:

other: Not siginificantly different from control

Species:

Triticum aestivum

Duration:

21 d

Dose descriptor:

other: % inhibition germination (from control)

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

element (total fraction)

Remarks:

ZnCl2

Basis for effect:

germination

Remarks:

% inhibition

Remarks on result:

other: Not siginificantly different from control

Species:

Triticum aestivum

Duration:

21 d

Dose descriptor:

other: % inhibition germination (from control)

Effect conc.:

250 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

element (total fraction)

Remarks:

ZnCl2

Basis for effect:

germination

Remarks:

% inhibition

Remarks on result:

other: Not siginificantly different from control

Species:

Raphanus sativus

Duration:

21 d

Dose descriptor:

other: % inhibition germination (from control)

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

element (total fraction)

Remarks:

ZnO nano

Basis for effect:

germination

Remarks:

% inhibition

Remarks on result:

other: Not siginificantly different from control

Species:

Raphanus sativus

Duration:

21 d

Dose descriptor:

other: % inhibition germination (from control)

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

element (total fraction)

Remarks:

ZnO bulk

Basis for effect:

germination

Remarks:

% inhibition

Remarks on result:

other: Not siginificantly different from control

Species:

Raphanus sativus

Duration:

21 d

Dose descriptor:

other: % inhibition germination (from control)

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

element (total fraction)

Remarks:

ZnCl2

Basis for effect:

germination

Remarks:

% inhibition

Remarks on result:

other: Not siginificantly different from control

Species:

Raphanus sativus

Duration:

21 d

Dose descriptor:

other: % inhibition germination (from control)

Effect conc.:

250 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

element (total fraction)

Remarks:

ZnCl2

Basis for effect:

germination

Remarks:

%inhibition

Remarks on result:

other: Not siginificantly different from control

Species:

Vicia sativa

Duration:

21 d

Dose descriptor:

other: % inhibition germination (from control)

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

element (total fraction)

Remarks:

ZnO nano

Basis for effect:

germination

Remarks:

% inhibition

Remarks on result:

other: Not siginificantly different from control

Species:

Vicia sativa

Duration:

21 d

Dose descriptor:

other: % inhibition germination (from control)

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

element (total fraction)

Remarks:

ZnO bulk

Basis for effect:

germination

Remarks:

% inhibition

Remarks on result:

other: Not siginificantly different from control

Species:

Vicia sativa

Duration:

21 d

Dose descriptor:

other: % inhibition germination (from control)

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

element (total fraction)

Remarks:

ZnCl2

Basis for effect:

germination

Remarks:

% inhibition

Remarks on result:

other: 50%

Species:

Vicia sativa

Duration:

21 d

Dose descriptor:

other: % inhibition germination (from control)

Effect conc.:

250 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

element (total fraction)

Remarks:

ZnCl2

Basis for effect:

germination

Remarks:

% inhibition

Remarks on result:

other: Not significantly different from control

Species:

Triticum aestivum

Duration:

21 d

Dose descriptor:

other: % inhibition root length (from control)

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

element (total fraction)

Remarks:

ZnO nano

Basis for effect:

growth

Remarks:

root length % inhibition

Remarks on result:

other: Not significantly different from control

Species:

Triticum aestivum

Duration:

21 d

Dose descriptor:

other: % inhibition root length (from control)

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

element (total fraction)

Remarks:

ZnO bulk

Basis for effect:

growth

Remarks:

root length % inhibition

Remarks on result:

other: Not significantly different from control

Species:

Triticum aestivum

Duration:

21 d

Dose descriptor:

other: % inhibition root length (from control)

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

element (total fraction)

Remarks:

ZnCl2

Basis for effect:

growth

Remarks:

root length % inhibition

Remarks on result:

other: 37%

Species:

Triticum aestivum

Duration:

21 d

Dose descriptor:

other: % inhibition root length (from control)

Effect conc.:

250 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

element (total fraction)

Remarks:

ZnCl2

Basis for effect:

growth

Remarks:

root length % inhibition

Remarks on result:

other: Not significantly different from control

Species:

Raphanus sativus

Duration:

21 d

Dose descriptor:

other: % inhibition root length (from control)

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

element (total fraction)

Remarks:

ZnO nano

Basis for effect:

growth

Remarks:

root length % inhibition

Remarks on result:

other: Not significantly different from control

Species:

Raphanus sativus

Duration:

21 d

Dose descriptor:

other: % inhibition root length (from control)

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

element (total fraction)

Remarks:

ZnO bulk

Basis for effect:

growth

Remarks:

root length % inhibition

Remarks on result:

other: Not significantly different from control

Species:

Raphanus sativus

Duration:

21 d

Dose descriptor:

other: % inhibition root length (from control)

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

element (total fraction)

Remarks:

ZnCl2

Basis for effect:

growth

Remarks:

root length % inhibition

Remarks on result:

other: 37%

Species:

Raphanus sativus

Duration:

21 d

Dose descriptor:

other: % inhibition root length (from control)

Effect conc.:

250 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

element (total fraction)

Remarks:

ZnCl2

Basis for effect:

growth

Remarks:

root length % inhibition

Remarks on result:

other: Not significantly different from control

Species:

Vicia sativa

Duration:

21 d

Dose descriptor:

other: % inhibition root length (from control)

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

element (total fraction)

Remarks:

ZnO nano

Basis for effect:

growth

Remarks:

root length % inhibition

Remarks on result:

other: Not significantly different from control

Species:

Vicia sativa

Duration:

21 d

Dose descriptor:

other: % inhibition root length (from control)

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

element (total fraction)

Remarks:

ZnO bulk

Basis for effect:

growth

Remarks:

root length % inhibition

Remarks on result:

other: Not significantly different from control

Species:

Vicia sativa

Duration:

21 d

Dose descriptor:

other: % inhibition root length (from control)

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

element (total fraction)

Remarks:

ZnCl2

Basis for effect:

growth

Remarks:

root length % inhibition

Remarks on result:

other: 48%

Species:

Vicia sativa

Duration:

21 d

Dose descriptor:

other: % inhibition root length (from control)

Effect conc.:

250 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

element (total fraction)

Remarks:

ZnCl2

Basis for effect:

growth

Remarks:

root length % inhibition

Remarks on result:

other: Not significantly different from control

Species:

Triticum aestivum

Duration:

21 d

Dose descriptor:

other: % inhibition shoot length (from control)

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

element (total fraction)

Remarks:

ZnO nano

Basis for effect:

growth

Remarks:

shoot length % inhibition

Remarks on result:

other: Not significantly different from control

Species:

Triticum aestivum

Duration:

21 d

Dose descriptor:

other: % inhibition shoot length (from control)

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

element (total fraction)

Remarks:

ZnO bulk

Basis for effect:

growth

Remarks:

shoot length % inhibition

Remarks on result:

other: Not significantly different from control

Species:

Triticum aestivum

Duration:

21 d

Dose descriptor:

other: % inhibition shoot length (from control)

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

element (total fraction)

Remarks:

ZnCl2

Basis for effect:

growth

Remarks:

shoot length % inhibition

Remarks on result:

other: 20%

Species:

Triticum aestivum

Duration:

21 d

Dose descriptor:

other: % inhibition shoot length (from control)

Effect conc.:

250 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

element (total fraction)

Remarks:

ZnCl2

Basis for effect:

growth

Remarks:

shoot length % inhibition

Remarks on result:

other: Not significantly different from control

Species:

Raphanus sativus

Duration:

21 d

Dose descriptor:

other: % inhibition shoot length (from control)

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

element (total fraction)

Remarks:

ZnO nano

Basis for effect:

growth

Remarks:

shoot length % inhibition

Remarks on result:

other: Not significantly different from control

Species:

Raphanus sativus

Duration:

21 d

Dose descriptor:

other: % inhibition shoot length (from control)

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

element (total fraction)

Remarks:

ZnO bulk

Basis for effect:

growth

Remarks:

shoot length % inhibition

Remarks on result:

other: Not significantly different from control

Species:

Raphanus sativus

Duration:

21 d

Dose descriptor:

other: % inhibition shoot length (from control)

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

element (total fraction)

Remarks:

ZnCl2

Basis for effect:

growth

Remarks:

shoot length % inhibition

Remarks on result:

other: 31%

Species:

Raphanus sativus

Duration:

21 d

Dose descriptor:

other: % inhibition shoot length (from control)

Effect conc.:

250 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

element (total fraction)

Remarks:

ZnCl2

Basis for effect:

growth

Remarks:

shoot length % inhibition

Remarks on result:

other: Not significantly different from control

Species:

Vicia sativa

Duration:

21 d

Dose descriptor:

other: % inhibition shoot length (from control)

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

element (total fraction)

Remarks:

ZnO nano

Basis for effect:

growth

Remarks:

shoot length % inhibition

Remarks on result:

other: 15%

Species:

Vicia sativa

Duration:

21 d

Dose descriptor:

other: % inhibition shoot length (from control)

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

element (total fraction)

Remarks:

ZnO bulk

Basis for effect:

growth

Remarks:

shoot length % inhibition

Remarks on result:

other: 12%

Species:

Vicia sativa

Duration:

21 d

Dose descriptor:

other: % inhibition shoot length (from control)

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

element (total fraction)

Remarks:

ZnCl2

Basis for effect:

growth

Remarks:

shoot length % inhibition

Remarks on result:

other: 32%

Species:

Vicia sativa

Duration:

21 d

Dose descriptor:

other: % inhibition shoot length (from control)

Effect conc.:

250 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

element (total fraction)

Remarks:

ZnCl2

Basis for effect:

growth

Remarks:

shoot length % inhibition

Remarks on result:

other: 11%

Species:

Triticum aestivum

Duration:

21 d

Dose descriptor:

other: % inhibition shoot weight (from control)

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

element (total fraction)

Remarks:

ZnO nano

Basis for effect:

growth

Remarks:

shoot weight % inhibition

Remarks on result:

other: Not significantly different from control

Species:

Triticum aestivum

Duration:

21 d

Dose descriptor:

other: % inhibition shoot weight (from control)

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

element (total fraction)

Remarks:

ZnO bulk

Basis for effect:

growth

Remarks:

shoot weight % inhibition

Remarks on result:

other: Not significantly different from control

Species:

Triticum aestivum

Duration:

21 d

Dose descriptor:

other: % inhibition shoot weight (from control)

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

element (total fraction)

Remarks:

ZnCl2

Basis for effect:

growth

Remarks:

shoot weight % inhibition

Remarks on result:

other: 32%

Species:

Triticum aestivum

Duration:

21 d

Dose descriptor:

other: % inhibition shoot weight (from control)

Effect conc.:

250 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

element (total fraction)

Remarks:

ZnCl2

Basis for effect:

growth

Remarks:

shoot weight % inhibition

Remarks on result:

other: Not significantly different from control

Species:

Raphanus sativus

Duration:

21 d

Dose descriptor:

other: % inhibition shoot weight (from control)

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

element (total fraction)

Remarks:

ZnO nano

Basis for effect:

growth

Remarks:

shoot weight % inhibition

Remarks on result:

other: 38%

Species:

Raphanus sativus

Duration:

21 d

Dose descriptor:

other: % inhibition shoot weight (from control)

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

element (total fraction)

Remarks:

ZnO bulk

Basis for effect:

growth

Remarks:

shoot weight % inhibition

Remarks on result:

other: 40%

Species:

Raphanus sativus

Duration:

21 d

Dose descriptor:

other: % inhibition shoot weight (from control)

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

element (total fraction)

Remarks:

ZnCl2

Basis for effect:

growth

Remarks:

shoot weight % inhibition

Remarks on result:

other: 70%

Species:

Raphanus sativus

Duration:

21 d

Dose descriptor:

other: % inhibition shoot weight (from control)

Effect conc.:

250 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

element (total fraction)

Remarks:

ZnCl2

Basis for effect:

growth

Remarks:

shoot weight % inhibition

Remarks on result:

other: Not significantly different from control

Species:

Vicia sativa

Duration:

21 d

Dose descriptor:

other: % inhibition shoot weight (from control)

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

element (total fraction)

Remarks:

ZnO nano

Basis for effect:

growth

Remarks:

shoot weight % inhibition

Remarks on result:

other: 25%

Species:

Vicia sativa

Duration:

21 d

Dose descriptor:

other: % inhibition shoot weight (from control)

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

element (total fraction)

Remarks:

ZnO bulk

Basis for effect:

growth

Remarks:

shoot weight % inhibition

Remarks on result:

other: Not significantly different from control

Species:

Vicia sativa

Duration:

21 d

Dose descriptor:

other: % inhibition shoot weight (from control)

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

element (total fraction)

Remarks:

ZnCl2

Basis for effect:

growth

Remarks:

shoot weight % inhibition

Remarks on result:

other: 45%

Species:

Vicia sativa

Duration:

21 d

Dose descriptor:

other: % inhibition shoot weight (from control)

Effect conc.:

250 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

element (total fraction)

Remarks:

ZnCl2

Basis for effect:

growth

Remarks:

shoot weight % inhibition

Remarks on result:

other: Not significantly different from control

Reported statistics and error estimates:

The data were analyzed statistically using the STAT GRAPHICS software (Version 5.0). Statistically significant differences between individual means for chemical and toxicological data were identified by one-way analysis of variance (ANOVA) with Fisher’s least significant difference procedure (LSD, p<0.05).

Total Zn uptake was calculated as the product of dry matter production and the total Zn concentration in roots or shoots. The transference factor (Tf) was defined as the ratio of the Zn concentration in shoots to that in roots. The bioconcentration factor (BCF) was calculated as the ratio of the Zn concentration measured in roots or shoots to that of the Zn in soil. Zinc concentrations are reported on a DW basis.

Validity criteria fulfilled:

yes

Conclusions:

Good quality study useful for assessing terrestrial toxicity.

Executive summary:

Toxicity of ZnO nanoparticles, ZnO bulk, and ZnCl2 (1000 mg Zn kg−1) was studied in wheat, radish, and vetch (emergence, root elongation, and shoot growth) in a natural soil. ZnCl2 presented the highest toxicity; ZnO-NPs and ZnO bulk effects were comparable.

Reference 15

Endpoint:

toxicity to terrestrial plants: long-term

Type of information:

experimental study

Adequacy of study:

weight of evidence

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:

The desired amounts of ZnO NPs were suspended in MPW and sonicated for 15min in awater bath (Crest Ultrasonics, Trenton, NJ). The NP suspensions were then applied to the soil to achieve concentrations of 250, 500, and 750 mg/kg. Control (without NPs) treatments were set up for all three different set of treatments. The pots were filled with 300 g of soil amended with desired amounts of nano ZnO, bulk ZnO, and ZnCl2. A control treatment (no Zn amendment), with its respective replicates, was also set up. The seeds were sown 24 h after soil amendment. Three replicates were prepared for each treatment.

GLP compliance:

not specified

Remarks:

GLP compliance not specified in publication

Specific details on test material used for the study:

ZnONPs (MeliorumTechnologies, Rochester, NY) of 10 nmmean diameter were obtained fromthe University of California Center for Environmental Implications of Nanotechnology (UC-CEIN). Three concentrations (250, 500, and 750 mg/kg of soil) of bulk ZnO and ZnCl2 (Sigma-Aldrich) were prepared simultaneously with the NPs in MPWand mixed with the soil.

Analytical monitoring:

no

Vehicle:

no

Details on preparation and application of test substrate:

The pots were filled with 300 g of soil amended with desired amounts of nano ZnO, bulk ZnO, and ZnCl2. A control treatment (no Zn amendment), with its respective replicates, was also set up. The seeds were sown 24 h after soil amendment. Three replicates were prepared for each treatment. Growth test went on for 30 days.

Species:

other: Medicago sativa

Plant group:

Dicotyledonae (dicots)

Details on test organisms:

Alfalfa (M. saliva L.) seeds (Del Norte Seed & Feed, Vinton, TX, USA) were sterilized in 4% sodium hypochlorite solution for 10 min and rinsed with MPW four times. Seeds were inoculated with S. melilotisuspension for 2 h before sowing into the soil.

Test type:

seedling emergence and seedling growth test

Study type:

laboratory study

Substrate type:

natural soil

Limit test:

no

Total exposure duration:

30 d

Test temperature:

25/20°C day/night temperature

Moisture:

65% relative humidity

Details on test conditions:

light intensity of 340 μmol s−1 m−2

Nominal and measured concentrations:

250, 500, and 750 mg/kg of soil

Reference substance (positive control):

no

Species:

other: Medicago sativa L.

Duration:

5 d

Dose descriptor:

LOEC

Effect conc.:

<= 500 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

element (total fraction)

Remarks:

from nano ZnO

Basis for effect:

germination

Species:

other: Medicago sativa L.

Duration:

5 d

Dose descriptor:

LOEC

Effect conc.:

<= 500 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

element (total fraction)

Remarks:

from bulk ZnO

Basis for effect:

germination

Species:

other: Medicago sativa L.

Duration:

5 d

Dose descriptor:

LOEC

Effect conc.:

<= 250 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

element (total fraction)

Remarks:

from ZnCl2

Basis for effect:

germination

Species:

other: Medicago sativa L.

Duration:

30 d

Dose descriptor:

LOEC

Effect conc.:

<= 250 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

element (total fraction)

Basis for effect:

growth

Remarks:

root dry biomass

Remarks on result:

other: same LOEC value for nano ZnO, bulk ZnO and ZnCl2

Species:

other: Medicago sativa L.

Duration:

30 d

Dose descriptor:

LOEC

Effect conc.:

<= 250 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

element (total fraction)

Basis for effect:

growth

Remarks:

shoot dry biomass

Remarks on result:

other: same LOEC values for nano ZnO and ZnCl2

Remarks:

bulk ZnO had a growth enhancing effect

Details on results:

Ratios between ZnO nano and zinc ion were calculated when an effect was seen in one of the two relative to control.

Reported statistics and error estimates:

Each concentration was set in triplicate including the control in a completely randomdesign. Separate sets of plantswere used for Zn concentration determination, physiologicalmeasurements, and microscopy imaging. The data (means ± SD) were reported as averages of three replicates. One-way ANOVA test was performed and Tukey-HSD multiple comparisons test performed using the statistical package SPSS version
12.0 (SPSS, Chicago, IL). Statistical significance was based on probabilities of p ≤ 0.05.

Validity criteria fulfilled:

yes

Conclusions:

This study does not follow guidelines but is usable as weight of evidence as it gives information on germination and growth.

Executive summary:

ZnO NPs did not affect germination rates at 250 and 500 mg/kg. However, the germination rate increased to 23% at 750 mg/kg ZnO NP treatment, compared to control. Bulk ZnO treatments showed 50% lower germination rate at 500 and 750 mg/kg compared to control. The ionic treatments were found to be germination inhibitors with higher ZnCl2 concentrations.

Results showed that ZnCl2 was phytotoxic at all concentrations, whereas ZnO NPs at 500 and 750 mg/kg treatments reduced plant growth and dry biomass production. Conversely, at 500 mg/kg bulk ZnO acted as alfalfa's growth promoter, as ZnO triggered the shoot dry biomass production and enhanced leaf area index.

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.