Zinc oxide

Toxicity to soil macroorganisms except arthropods

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to soil macroorganisms except arthropods: long-term

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: No guideline followed and no comparison to bulk and soluble zinc compound

Qualifier:

no guideline followed

Principles of method if other than guideline:

Age synchronous populations of L1–larval nematodes were obtained by the collection. Exposures to different metal oxide NPs at the
examined concentrations were performed from L1–larval stage in 12-well sterile tissue culture plates at 20 C incubator in the presence
of food, and the exposed nematodes were used for toxicity evaluation using lethality, growth, locomotion behavior and reproduction as endpoints when they developed into adults

GLP compliance:

no

Specific details on test material used for the study:

The sizes of all used metal oxide NPs were 30 nm without any coating throughout this study. ZnO–NPs were from Nano Applied Research Center of Nanjing University of Technology. Purities of ZnO–NPs were >99%. Shapes of the used NPs were determined using a transmission electron microscope (TEM) (JEM-100CXII, JEOL, Ltd, Japan). Zeta-potentials were 0.32 mV. Wide distributions of particle sizes of ZnO–NPs were 627 ± 175 nm, which
were determined by a Nano-Zetasizer (1000 HS, Malvern Instrument Ltd., UK) using a dynamic light scattering (DLS) technique.

Analytical monitoring:

yes

Vehicle:

no

Test organisms (species):

Caenorhabditis elegans

Animal group:

nematods

Details on test organisms:

Nematodes used in the present study were wild-type N2, originally obtained from the Caenorhabditis Genetics Center (funded by the NIH National Center for Research Resource, USA), which were maintained on nematode growth medium (NGM) plates seeded with Escherichia coli OP50 at 20 C as described (Brenner, 1974). Gravid nematodes were washed off the plates into centrifuge tubes, and were lysed with a bleaching mixture (0.45 M NaOH,
2% HOCl). Age synchronous populations of L1–larval nematodes were obtained by the collection as described (Donkin and Dusenbery, 1993). Exposures to different metal oxide NPs at the examined concentrations were performed from L1–larval stage in 12-well sterile tissue culture plates at 20 C incubator in the presence of food, and the exposed nematodes were used for toxicity evaluation using lethality, growth, locomotion behavior and reproduction as endpoints when they developed into adults.

Study type:

laboratory study

Substrate type:

other: culture plates

Remarks:

from juvenile until adult

Test temperature:

20°C

Nominal and measured concentrations:

0.0005, 0.005, 0.05, 0.5, 5, 10, and 50 µg/L

Dose descriptor:

NOEC

Effect conc.:

10 other: µg/L

Nominal / measured:

nominal

Conc. based on:

element (total fraction)

Remarks:

nZnO

Basis for effect:

mortality

Dose descriptor:

NOEC

Effect conc.:

10 other: µg/L

Nominal / measured:

nominal

Conc. based on:

element (total fraction)

Remarks:

nZnO

Basis for effect:

growth

Remarks:

body length

Dose descriptor:

NOEC

Effect conc.:

0.05 other: µg/L

Nominal / measured:

nominal

Conc. based on:

element (total fraction)

Remarks:

nZnO

Basis for effect:

reproduction

Remarks:

body size

Reported statistics and error estimates:

All data were expressed as means ± standard error of the mean (S.E.M.). Statistical analysis was performed using SPSS 12.0 (SPSS Inc., Chicago, IL, USA). Analysis of variance (ANOVA) was used to determine the significance of differences between the groups.
Probability levels of 0.05 and 0.01 were considered statistically significant. Associations of ROS production with lethality, growth, reproduction and locomotion behavior were assessed with linear regression analysis. The dependent variables were lethality, growth, reproduction and locomotion behavior, and the independent variable was ROS production.

Validity criteria fulfilled:

no

Conclusions:

no guideline followed and no comparison to bulk form or ion, thus considered as supportive only

Reference 2

Endpoint:

toxicity to soil macroorganisms except arthropods: long-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: study not according to standard protocol , not relevant for soil toxicity since done in water ; other conditions also irrelevant for soil environment

Qualifier:

no guideline followed

Principles of method if other than guideline:

All the tests were carried out in ultrapure water and no salts were added. At the beginning of each test, approximately 30 L1 juveniles were transferred by micropipette to the Petri plates containing different concentrations of toxicity test particle (i.e., nanoparticulate and bulk ZnO) After 5d the surviving worms were counted with a dissecting microscope. Death was determined as a lack of response to gentle probing with a needle.The length and reproduction were determined.

GLP compliance:

not specified

Analytical monitoring:

yes

Details on sampling:

A total of 40 mL of each concentration of NP suspensions was centrifuged at 13,000 g for 20 min and filtered (0.2 mm polytetrafluoroethylene filter, Ireland). To determine dissolved metal (defined as metal present in the supernatant), 6 mL of the supernatant was acidified with 250 mL of 1% HNO3 (V:V) and analyzed by an inductively coupled plasma mass spectrometer (Elan 6100, PerkinElmer, USA). The remaining supernatant was also tested for the same four endpoints to elucidate if the toxicity was caused by the particles or the dissolved metal ions. In addition, the time- and concentration-dependent dissolution of nanoparticulate and bulk particles was also investigated using the above procedure.

Vehicle:

no

Test organisms (species):

Caenorhabditis elegans

Animal group:

nematods

Details on test organisms:

C. elegans strain Bristol N2 (wild type) used in this work was provided by the Caenorhabditis Genetics Center (University of Minnesota) and maintained on nematode growth medium (NGM) plates (95 mm 15 mm sterilized-disposable Petri dish, Canada) seeded with E. coli strain OP50 at 201 C

Study type:

laboratory study

Substrate type:

other: aqueous media

Limit test:

no

Total exposure duration:

5 d

Test temperature:

20°C

pH:

7

Nominal and measured concentrations:

0.4, 0.8, 1.6, 4.1, 6.1 and 8.1 mg/l

Reference substance (positive control):

no

Duration:

5 d

Dose descriptor:

NOEC

Effect conc.:

0.8 other: mg/l

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

nZnO

Basis for effect:

growth

Duration:

5 d

Dose descriptor:

NOEC

Effect conc.:

0.8 other: mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

nZnO

Basis for effect:

reproduction

Remarks:

eggs inside body

Duration:

5 d

Dose descriptor:

NOEC

Effect conc.:

0.4 other: mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

nZnO

Basis for effect:

reproduction

Remarks:

offspring per worm

The measured diameter of ZnO bulk particles were 429 nm, much larger than their nano-sized counterparts. However,

considerable particle aggregation was observed by TEM for each of the investigated particles. Using DLS, wide distributions

of particle size were observed, from 478 to 980 nm for ZnO considerably larger than their nominal size 20, 60 and 50 nm, respectively. It should be noted that particle sizes measured by the Nano-Sizer are hydrodynamic diameter based on the Stokes–Einstein

equation, which are expected to be larger than the actual particle size. Also, the specific surface area of NPs was significantly

higher than that of bulk particles.

Validity criteria fulfilled:

yes

Conclusions:

Good quality test and considered useful for assessing chronic soil ecotoxicity.

Executive summary:

Effects of nZnO on C. elegans exposed for 5 days revealed that effect of ZnO NP could be partially related to the release of dissolved Zn2 +. Effects of nZnO were comparable with bulk ZnO for growth and number of eggs inside the worm. Only for offspring per worm the effect of nZnO was larger compared with bulk ZnO.

Test considered irrelevant for soil toxicity since done in water.

Reference 3

Endpoint:

toxicity to soil macroorganisms except arthropods: 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: study not according to standard protocol, not done in soil but in water

Qualifier:

equivalent or similar to guideline

Guideline:

other: ASTM E2172 - standard guide for conducting lab soil test with nematode C. elegans

Principles of method if other than guideline:

All the tests were carried out in ultrapure water and no salts were added. At the beginning of each test, approximately 30 L1 juveniles were transferred by micropipette to the Petri plates containing different concentrations of toxicity test particle (i.e., nanoparticulate and bulk ZnO). 250 mL of E. coli in LB broth
were centrifuged at 3000 g for 10 min, and the bacterial pellet was used as the concentrated bacteria to feed the nematodes. A loopful of the pelleted bacteria was transferred to the above test plates. We chose an aqueous medium because it was difficult to evenly distribute NPs in an agar medium and the nematodes were able to freely contact with NPs in the aqueous medium; also, C. elegans lives in the interstitial water of soil. The exact number of transferred L1 nematodeswas counted with a dissecting microscope. The seeded plates were incubated at 20 C under dark condition and shaken at 100 rpm. After 24 h the surviving worms were counted with a dissecting microscope. Death was determined as a lack of response to gentle probing with a needle.

GLP compliance:

not specified

Specific details on test material used for the study:

- Name of test material (as cited in study report): colloidal ZnO in aqueous suspension
- Physical state: solid, nanoparticles of 20nm diameter
- purity 99.5%
- Hydrodynamic diameter: 478-980nm
- zeta potential: 0.017
- surface area: 54 m²/g
- Analytical purity: no data

Analytical monitoring:

no

Vehicle:

no

Test organisms (species):

Caenorhabditis elegans

Animal group:

nematods

Study type:

laboratory study

Substrate type:

other: aqueous media

Limit test:

no

Total exposure duration:

24 h

Test temperature:

20°C

pH:

7

Nominal and measured concentrations:

0.4, 0.8, 1.6, 4.1, 6.1 and 8.1 mg/l

Reference substance (positive control):

no

Duration:

24 h

Dose descriptor:

LC50

Effect conc.:

2.3 other: mg/l

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Validity criteria fulfilled:

yes

Conclusions:

Good quality test and considered useful for assessing acute soil ecotoxicity.

Executive summary:

In this research we characterized the toxicity and behavior of nanoparticulate and bulk metal oxide ZnO C. elegans in an aqueous exposure medium. We have shown (1) that metal oxide NPs are toxic to C. elegans, especially to its reproductive capability; (2) and that this toxicity could not be adequately explained by dissolution of the particles alone. The present study has taken a first step in the direction of investigating the ecotoxicity of metal oxide NPs to C. elegans and highlights the need for integrated toxicological assessment of metal oxide NPs in soil and aquatic systems.

tests not considered relevant for soil toxicity since done in water.

Reference 4

Endpoint:

toxicity to soil macroorganisms except arthropods: 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:

Toxicity tests were conducted in 24-well microtiter plates. Each well used in the test contained 2 mL treating agent and ten nematodes. The experiments were repeated four times for all the treatments. At the end of the treatments, mortality was determined by establishing the number of dead nematodes for the given treatment. The death of the worms was studied under a transmission stereo microscope (Olympus SZH 10, Olympus Optical CO., LTD., Tokyo, Japan) and defined as a motionless state which cannot be changed by manipulating the animal with a plastic needle. Control samples were also investigated; the mortality was low—0 dead animals in most cases and sometimes 1.

GLP compliance:

not specified

Remarks:

GLP compliance not specified in the publication

Specific details on test material used for the study:

For the treatments of nematodes—beyond ZnSO4 solutions—bulk and nano-ZnO suspensions were applied. The characterization of both the powders and their suspensions was carried out before the toxicological investigations. Both types of ZnO powders were ordered from Sigma-Aldrich Ltd. (Budapest, Hungary). According to the specification of the nano-ZnO, its grain size amounted to ≤50 nm.

Vehicle:

no

Test organisms (species):

other: Xiphinema vuittenezi

Animal group:

nematods

Details on test organisms:

Females of the plant-feeding nematode, X. vuittenezi, were used throughout the whole experiment. The worms were collected from a garden in the outskirts of Budapest. Extraction technique was a modification of Cobb’s sieves.

Study type:

laboratory study

Substrate type:

other: solution

Limit test:

no

Total exposure duration:

24 h

Test temperature:

20+/-1°C

Nominal and measured concentrations:

5, 25, 50 mg Zn/l

Duration:

24 h

Dose descriptor:

LC100

Effect conc.:

> 5 - < 25 other: mg Zn/l

Nominal / measured:

meas. (initial)

Conc. based on:

element (dissolved fraction)

Remarks:

from nano ZnO

Basis for effect:

mortality

Duration:

24 h

Dose descriptor:

LC100

Effect conc.:

> 25 - < 50 other: mg Zn/l

Nominal / measured:

meas. (initial)

Conc. based on:

element (dissolved fraction)

Remarks:

from ZnSO4

Basis for effect:

mortality

Reported statistics and error estimates:

Average particle size in the bulk and nano-ZnO suspension was determined by the measurement of the size of 150 individual
particles using the pictures presented in Fig. 1a, b, respectively. Measurements were conducted using the ImageJ image processing software (National Institutes of Health, Bethesda, USA). Results are presented as mean±standard error of the mean. For the comparison of zinc content of living versus previously killed nematodes as well as zinc uptake from suspension containing versus not containing soil solution, two-sample t test was applied; significance level was set to p<0.05. In the case of potassium and zinc content of nematodes treated with bulk and nano-
ZnO suspension as well as ZnSO4 solution, one-way ANOVA and Tukey post hoc test were applied. Significance level was the same as in the case of the t tests. Toxicity and zinc uptake were expressed in the function of dissolved zinc as well. For 5 and 50 mg/L concentrations, dissolved zinc content was determined directly by the TXRF measurement described before; for 25 mg/ L concentration, it was calculated by interpolation.

Validity criteria fulfilled:

yes

Conclusions:

The mortality caused by nanoZnO and ZnSO4 is comparable.

Reference 5

Endpoint:

toxicity to soil macroorganisms except arthropods: 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: No guideline followed; test results not reliable since not done in soil but water; but comparison nano, bulk and ZnCl2

Qualifier:

no guideline followed

Principles of method if other than guideline:

Each test consisted of six concentrations of test substance (4, 8, 20, 40, 60, 80 mg/l Zn, corresponding to 5, 10, 25, 50, 75, 100 mg/l ZnO and 8, 17, 42, 84, 125, 167 mg/l ZnCl2) and a control, with three replicate wells for each concentration. A 1.0 ml aliquot of test solution was added to each well which was subsequently loaded with 10 (1) nematodes

GLP compliance:

not specified

Specific details on test material used for the study:

Powdered nanoparticulate ZnO (NanoGard zinc oxide) was purchased from Alfa Aesar (Ward Hill, MA, USA) with a stated size of 40e100 nm. Stock suspensions of nano-ZnO and bulk-ZnO (Mallinckrodt; Phillipsburg, NJ) (100 mg/l (80% Zn)) were prepared by sonication for 2 h in an ultrasonic bath (Branson; Danbury, CT). Specific surface areas (SSA) of both materials were determined by BrunauereEmmeteTeller (BET) method and were found to be 17.0 and 4.2 m2/g for nano-ZnO and bulk-ZnO, respectively. Reagent grade ZnCl2 (Mallinckrodt; Phillipsburg, NJ) was used to make ZnCl2 stock solution.

Analytical monitoring:

yes

Vehicle:

no

Test organisms (species):

Caenorhabditis elegans

Animal group:

nematods

Details on test organisms:

C. elegans (wild type N2) was obtained from Caenorhabditis Genetics Center (Minneapolis, MN).

Study type:

laboratory study

Substrate type:

other: nematode growth medium

Limit test:

no

Total exposure duration:

24 h

Test temperature:

25+/-1.5°C in natural sunlight
20+/-0.5°C in artificial light

Nominal and measured concentrations:

0, 20, 40, 60, 80, 100, 120 mg/l Zn

Duration:

2 h

Dose descriptor:

LC50

Effect conc.:

38 other: mg/l

Nominal / measured:

nominal

Conc. based on:

element

Remarks:

Zinc from nano ZnO

Basis for effect:

mortality

Remarks on result:

other: natural light

Duration:

2 h

Dose descriptor:

LC50

Effect conc.:

65 other: mg/l

Nominal / measured:

nominal

Conc. based on:

element

Remarks:

Zn from bulk ZnO

Basis for effect:

mortality

Remarks on result:

other: natural light

Duration:

2 h

Dose descriptor:

EC0

Effect conc.:

> 120 other: mg/l

Nominal / measured:

nominal

Conc. based on:

element

Remarks:

Zinc from ZnCl2

Basis for effect:

mortality

Remarks on result:

other: natural light

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

17 other: mg/l

Nominal / measured:

nominal

Conc. based on:

element

Remarks:

Zinc from nano ZnO

Basis for effect:

mortality

Remarks on result:

other: 2h natural light + 22h artificial light

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

38 other: mg/l

Nominal / measured:

nominal

Conc. based on:

element

Remarks:

Zinc from bulk ZnO

Basis for effect:

mortality

Remarks on result:

other: 2h natural light + 22h artificial light

Duration:

24 h

Dose descriptor:

EC0

Effect conc.:

> 120 other: mg/l

Nominal / measured:

nominal

Conc. based on:

element

Remarks:

Zinc from ZnCl2

Basis for effect:

mortality

Remarks on result:

other: 2h natural light + 22h artificial light

Details on results:

No mortality if exposure in the dark

Reported statistics and error estimates:

All data reported were based on three independent experiments. Median lethal concentrations (LC50s) and corresponding 95% CIs were calculated by Probit analysis using TOXSTAT software (WEST, Inc, 1994).

Validity criteria fulfilled:

yes

Conclusions:

No guideline followed but exploitable results for terrestrial toxicity

Executive summary:

Findings from the present study demonstrate that manufactured, nanosized ZnO particles can cause toxicological effects in the nematode C. elegans. Natural light induced increased toxicity than artificial light. Test results are not considered relevant for soil toxicity since done in water.

Reference 6

Endpoint:

toxicity to soil macroorganisms except arthropods: 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: No guideline followed ; comparison between nano-ZnO and ZnCl2, but results not considered useful for soil toxicity since done in water

Qualifier:

no guideline followed

Principles of method if other than guideline:

For the 24-h lethality test, exposure was conducted in 24-well tissue culture plates (Corning Costar). Each test consisted of five to six ZnO-NPs or ZnCl2 concentrations (range, 325– 1,625 mg Zn/L) and a control, with three replicate wells for each concentration and the control

GLP compliance:

not specified

Specific details on test material used for the study:

Pinnacle AF ZnO-NPs suspension, with a reported primary particle size of 2 to 6 nm, was purchased from Applied Nanoworks, Reagent-grade ZnCl2 (Sigma-Aldrich) was used, and various concentrations were made in both buffered and unbuffered K-medium for toxicity test.

Analytical monitoring:

yes

Vehicle:

no

Test organisms (species):

Caenorhabditis elegans

Animal group:

nematods

Details on test organisms:

The wild-type N2 strain of C. elegans originally was obtained from the Caenorhabditis Genetics Center and was maintained as a dauer larva stock in M9 buffer, replenished monthly [23]. All cultures were maintained at 20C. Four-day-old worms from age-synchronized cultures were prepared using the methods described by Donkin and Williams

Study type:

laboratory study

Substrate type:

other: nematode growth medium

Limit test:

no

Total exposure duration:

24 h

Test temperature:

20°C

Nominal and measured concentrations:

0, 400, 800, 1200, 1600, 2000 mg/l Zn

Reference substance (positive control):

yes

Remarks:

Cu

Duration:

24 h

Dose descriptor:

LC50

Effect conc.:

789 other: mg/l

Nominal / measured:

nominal

Conc. based on:

element

Remarks:

Zinc

Basis for effect:

mortality

Remarks on result:

other: nano ZnO

Duration:

24 h

Dose descriptor:

LC50

Effect conc.:

884 other: mg/l

Nominal / measured:

nominal

Conc. based on:

element

Remarks:

Zn

Basis for effect:

mortality

Remarks on result:

other: ZnCl2

Duration:

4 h

Dose descriptor:

EC50

Effect conc.:

635 other: mg/l

Nominal / measured:

nominal

Conc. based on:

element

Remarks:

Zinc

Basis for effect:

behaviour

Remarks:

movement

Remarks on result:

other: nano ZnO

Duration:

4 h

Dose descriptor:

EC50

Effect conc.:

546 other: mg/l

Nominal / measured:

nominal

Conc. based on:

element

Remarks:

Zinc

Basis for effect:

behaviour

Remarks:

movement

Remarks on result:

other: ZnCl2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

46 other: mg/l

Nominal / measured:

nominal

Conc. based on:

element

Remarks:

Zinc

Basis for effect:

reproduction

Remarks:

number of offsprings

Remarks on result:

other: nano ZnO

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

59 other: mg/l

Nominal / measured:

nominal

Conc. based on:

element

Remarks:

Zinc

Basis for effect:

reproduction

Remarks:

number of offsprings

Remarks on result:

other: ZnCl2

Validity criteria fulfilled:

yes

Conclusions:

No guideline followed but exploitable results for terrestrial toxicity

Executive summary:

Findings from the present study suggest that manufactured, nanosized ZnO particles can cause toxicological effects in the nematode C. elegans. Using a variety of endpoints, the nanoparticles were determined to have toxicity comparable to that of ZnCl2 to C. elegans.

Results are not considered useful for soil toxicity since done in water.

Reference 7

Endpoint:

toxicity to soil macroorganisms except arthropods: short-term

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Tests done according to standard protocol but no comparison with bulk or salt. Good quality but limited to LOEC value

Qualifier:

according to guideline

Guideline:

OECD Guideline 207 (Earthworm, Acute Toxicity Tests)

GLP compliance:

not specified

Specific details on test material used for the study:

ZnO NP powder was purchased from Nanjing Emperor Nano Material Co., Nanjing, China, with a purity of 99.9%. Nominal ranges of particle diameters as provided by the manufacturer were 30±5 nm. Particle size and morphology were characterized by an H-600 TEM (Hitachi, Tokyo, Japan)

Analytical monitoring:

yes

Vehicle:

no

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

The earthworms used in this study were pre-clitellate specimens of the species E. fetida. They were kept for several weeks in darkness in cow dung at a constant temperature of 25 °C in a climate-controlled room.

Study type:

laboratory study

Substrate type:

filter paper

Limit test:

no

Total exposure duration:

96 h

Test temperature:

25 °C

Nominal and measured concentrations:

0, 50, 100, 200, 500, and 1000 mg/L

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

LOEC

Effect conc.:

50 other: mg ZnO/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

ZnO

Basis for effect:

mortality

Remarks on result:

other: tested in deionized water

Reported statistics and error estimates:

Data including SOD, CAT, and GSH-Px activities are presented as the mean±SD. The differences between groups were tested for significance using one-way analysis of variance (ANOVA). Differences were considered significant at pb0.05. The 96-h median lethal concentrations (LC50-values) were calculated using the trimmed Spearman–Karber method (Hamilton et al., 1977).

In our experiment, the amount of zinc released from ZnO NPs in agar was measured. Our results indicated that the lowest exposure concentration of ZnO NPs (50 mg/kg agar) released the highest amount of zinc ions and the extent of particle solubility in agar prepared with DW is larger than in case of using RW. This result was in agreement with the above theories, possibly due to the considerable aggregation observed in agar prepared with RW. The formation of agglomerates hinders dissolution, which can be attributed to reduction of the average equilibrium solubility of the particle system and the introduction of kinetic hindrance in the diffusion process. Exactly how aggregation affects the dissolution behavior of particles was not well understood.

Validity criteria fulfilled:

yes

Conclusions:

Good quality study but limited to LOEC value

Executive summary:

The dispersion and solubility of ZnO NPs vary significantly with solution chemistry. Different effects of ZnO NPs on earthworms were

observed depending on the method of exposure employed. After short-term (96 h) exposure of E. fetida to ZnO NPs in agar, earthworm mortality increased with increasing concentrations. When the worms were exposed on filter paper, which only represents dermal uptake, toxicity decreased with increasing ZnO NP concentration. This study showed the potential of soil extracts to mitigate the toxic effects of ZnO NPs on filter paper, which may also be attributed to the presence of salts and organic carbon. The use of soil extract as test medium in ecotoxicological assays can increase the predictive power of such filter paper tests for the environmental risk assessment of NPs. In addition, the dissolution behavior and potential toxicity of ZnO NPs need to be considered to assess their specific risks, when behavior and transfer of NPs in the soil ecosystems are investigated.

Reference 8

Endpoint:

toxicity to soil macroorganisms except arthropods: 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:

Each NP treatment concentration was prepared separately, without dilution, by weighing particles and dispersing them in agar.
ZnO NP concentrations of 0, 50, 100, 200, 500, and 1000 mg/kg were prepared in three replicate test units per treatment. Agar media without NPs were used as the negative control. Six randomly selected worms were placed just on the surface of the agar in the test units. After 96 h exposure in the agarose medium.

GLP compliance:

not specified

Remarks:

GLP compliance not specified in publication

Specific details on test material used for the study:

ZnO NP powder was purchased from Nanjing Emperor Nano Material Co., Nanjing, China, with a purity of 99.9%. Nominal ranges of particle diameters as provided by the manufacturer were 30±5 nm. Particle size and morphology were characterized by an H-600 TEM (Hitachi, Tokyo, Japan)

Analytical monitoring:

yes

Vehicle:

no

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

The earthworms used in this study were pre-clitellate specimens of the species E. fetida. They were kept for several weeks in darkness in cow dung at a constant temperature of 25 °C in a climate-controlled room.

Study type:

laboratory study

Substrate type:

other: agar

Limit test:

no

Total exposure duration:

96 h

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

232 other: mg ZnO/kg agar

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

ZnO

Basis for effect:

mortality

Remarks on result:

other: tested in deionized water

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

374 other: mg ZnO/kg agar

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

ZnO

Basis for effect:

mortality

Remarks on result:

other: tested in reconsituted water

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

1.4 other: mg Zn/kg agar

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

Zn

Basis for effect:

mortality

In our experiment, the amount of zinc released from ZnO NPs in agar was measured. Our results indicated that the lowest exposure concentration of ZnO NPs (50 mg/kg agar) released the highest amount of zinc ions and the extent of particle solubility in agar prepared with DW is larger than in case of using RW. This result was in agreement with the above theories, possibly due to the considerable aggregation observed in agar prepared with RW. The formation of agglomerates hinders dissolution, which can be attributed to reduction of the average equilibrium solubility of the particle system and the introduction of kinetic hindrance in the diffusion process. Exactly how aggregation affects the dissolution behavior of particles was not well understood.

Validity criteria fulfilled:

yes

Conclusions:

Study useful for assessing terretrial toxicity

Executive summary:

The dispersion and solubility of ZnO NPs vary significantly with solution chemistry. Different effects of ZnO NPs on earthworms were

observed depending on the method of exposure employed. After short-term (96 h) exposure of E. fetida to ZnO NPs in agar, earthworm mortality increased with increasing concentrations. When the worms were exposed on filter paper, which only represents dermal uptake, toxicity decreased with increasing ZnO NP concentration. This study showed the potential of soil extracts to mitigate the toxic effects of ZnO NPs on filter paper, which may also be attributed to the presence of salts and organic carbon. The use of soil extract as test medium in ecotoxicological assays can increase the predictive power of such filter paper tests for the environmental risk assessment of NPs. In addition, the dissolution behavior and potential toxicity of ZnO NPs need to be considered to assess their specific risks, when behavior and transfer of NPs in the soil ecosystems are investigated.

Reference 9

Endpoint:

toxicity to soil macroorganisms except arthropods: long-term

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Qualifier:

no guideline followed

Principles of method if other than guideline:

Populations of eight worms were exposed to a control and two concentrations of both NP ZnO and ZnCl2 (Sigma-Aldrich) dosed to soil and food for 21 d. Estimated ‘low’ and ‘high’ Zn concentrations 250 and 750 mg kg−1 respectively.

GLP compliance:

not specified

Remarks:

GLP compliance not specified in publication

Specific details on test material used for the study:

Uncoated ZnO nanopowder with a stated particle diameter in the nanoparticulate range of b100 nm and a BET calculated surface area of 15–25 m2 g−1 was obtained from Sigma-Aldrich (Poole, UK).

Analytical monitoring:

no

Vehicle:

no

Test organisms (species):

Eisenia sp.

Animal group:

annelids

Details on test organisms:

The earthworm Eisenia veneta (Rosa, 1886) was obtained from a sheltered outdoor culture which wasmaintained in a reconstituted soil made up of 33% loamy top soil (Madingly Mulch, Cambridge, UK), 33% Sphagnumpeat, and 33% composted bark (both LBS Horticultural, Colne, UK). Worm cultures were kept moist using a trickle watering system and fed liberally with horse manure, which was frozen before use. A week before the experimental exposures, E. veneta adults with a well developed clitellum were removed from the culture and maintained in the laboratory in culture soil mediumunder the defined tests conditions of 12±1.5 °C in a 16 h L: 8 h D photoperiod. These conditions were designed to be representative to average spring/autumn conditions in a temperature soil.

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

21 d

Test temperature:

12°C +- 1.5°C

Details on test conditions:

Populations of eight worms with an average individual weight of 0.92 g (SD=0.084)were exposed to a control and two concentrations of both NP ZnO and ZnCl2 (Sigma-Aldrich) dosed to soil and food for 21 d. Estimated ‘low’ and ‘high’ Zn concentrations (250 and 750 mg kg−1, respectively. Replicate (n=3) populations were exposed to each treatment in a 2 L plastic containerwith a perforated lid and 1.47 kg (dry wt) of soil. The test soil consisted of a commercially available clay loam (Broughton Loam, Kettering, UK) amended with 3% organic matter as composted bark, with a pH of 7.1 and total organic matter of 5% .

Nominal and measured concentrations:

nominal: 0-250-750 mg/kg dw

Reference substance (positive control):

no

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

750 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

element

Remarks:

Zinc

Basis for effect:

mortality

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

750 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

element

Remarks:

Zinc

Basis for effect:

other: cocoon production

Details on results:

After 21 d exposure, all adult worms survived except one individual at 750 mg kg−1 of ZnCl2. In all replicates, population weights increased during the 21 d test. Whilst within the GLM model, Zn concentration alone had no significant effect (PN0.05), the effect of Zn type (nanoparticle or ionic) was significant (F1,8=5.42, P=0.048). This effectwas best illustrated by the response at 750 mg kg−1where compared to the control, the change in weight was ~5% greater than in controls when exposed to NP ZnO, but ~5% lowerwhen exposed to ZnCl2 (Fig. 2a) (although the latter was possibly bias by the single mortality). The interaction between Zn types and exposure concentration was not, however, significant (P=0.075). Both Zn concentration (F1,8=6.32, P=0.036) and Zn type (F1,8=7.43, P=0.026) had an effect on cocoon production rate. Fewer cocoons were produced at 750 mg kg−1 for both Zn types than in the control soils. This effect was most pronounced for populations exposed to ZnCl2.

Validity criteria fulfilled:

yes

Executive summary:

Eisenia veneta were exposed for 21 days at 0 -250 -750 mg/kg nZnO or ZnCl2. The effects of nZnO were lower than observed for ZnCl2 at the same concentrations.

Reference 10

Endpoint:

toxicity to soil macroorganisms except arthropods: short-term

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: No guideline followed, exposure not in soil but in water

Qualifier:

no guideline followed

Principles of method if other than guideline:

Individual adultworms (n=10), which were not starved to avoid pre-exposure stress, were exposed to five increasing concentrations (6 to 96 mg Zn L−1) of NP Zn in 100mL of DI water for 24 h at 12 °C in constant darkness.

GLP compliance:

not specified

Specific details on test material used for the study:

Uncoated ZnO nanopowder with a stated particle diameter in the nanoparticulate range of <100 nm and a BET calculated surface area of 15–25 m2 g−1 was obtained from Sigma-Aldrich (Poole, UK).

Analytical monitoring:

yes

Details on sampling:

Water samples (10 mL) were obtained from the water column of two replicates of each treatment
immediately before adding theworms and at the end of the test. Samples were immediately frozen and later prepared by either microwave
digestion in 10% nitric acid for NP ZnO or acidified with 1% nitric acid for ZnCl2 before analysis for total Zn using ICP-OES (Perkin Elmer DV
4300).

Vehicle:

no

Test organisms (species):

other: Eisenia veneta

Animal group:

annelids

Details on test organisms:

The earthworm Eisenia veneta (Rosa, 1886) was obtained from a sheltered outdoor culture which wasmaintained in a reconstituted soil made up of 33% loamy top soil (Madingly Mulch, Cambridge, UK), 33% Sphagnumpeat, and 33% composted bark (both LBS Horticultural, Colne, UK). Worm cultures were kept moist using a trickle watering system and fed liberally with horse manure, which was frozen before use. A week before the experimental exposures, E. veneta adults with a well developed clitellum were removed from the culture and maintained in the laboratory in culture soil mediumunder the defined tests conditions of 12±1.5 °C in a 16 h L: 8 h D photoperiod. These conditions were designed to be representative to average spring/autumn conditions in a temperature soil.

Study type:

laboratory study

Substrate type:

other: DI water

Limit test:

no

Total exposure duration:

24 h

Nominal and measured concentrations:

nominal:6-96 mg Zn/L

Reference substance (positive control):

no

Duration:

24 h

Dose descriptor:

LC50

Effect conc.:

6.5 other: mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Remarks:

Zinc

Basis for effect:

mortality

Remarks on result:

other: ZnCl2

Duration:

24 h

Dose descriptor:

LC50

Effect conc.:

1.75 other: mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

element

Remarks:

Zn

Basis for effect:

mortality

Remarks on result:

other: nano ZnO

Reported statistics and error estimates:

The measured effects of NP ZnO and ZnCl2 were analysed using a general linearmodel (GLM) followed by Tukey's pair-wise comparisons (Minitab 15). Two factors, fixed and crossed (i.e. Zn type, nominal Zn concentration, Zn type×nominal Zn concentration) were specified for the model. Prior to analysis, data were transformed (square root for body concentrations and cocoon production rate; arcsine square root for immune activity and body wt) to comply with the assumptions of the GLM. Lethal concentrations (LC50) were estimated for the 24 h immersion test using Probit analysis based on the log10 transformed Zn concentrations, which gave adequate model fits (Minitab 15).

Validity criteria fulfilled:

no

Conclusions:

Tests not done according to standard protocol, exposure not in soil but in water. test considered supportive for checking the effect of nano-ZnO as compared to the bulk-ZnO form or the soluble ion.

Executive summary:

The data presented here represent a structured assessment of the chronic effects of NP ZnO and the mass equivalent Zn2+ ion to earthworms in a soil matrix. Our results indicated a lower toxicity of NP ZnO on reproduction and immune activity than ZnCl2 in E. veneta even though both forms reached similar body burdens at the same exposure concentration. In terms of risk assessment, our findings suggest that for initial analysis there is no apparent need to go beyond considering the metal component of larger size uncoated ZnO NPs in a soil matrix.

Reference 11

Endpoint:

toxicity to soil macroorganisms except arthropods: long-term

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 207 (Earthworm, Acute Toxicity Tests)

Deviations:

yes

Remarks:

natural soil, longer exposure

GLP compliance:

not specified

Remarks:

GLP not specified in the publication

Specific details on test material used for the study:

Uncoated ZnO-NP powder (advertised particle size\100-nm diameter) and the bulk form of ZnO were purchased from Sigma-Aldrich (Germany), and ZnCl2 salt was purchased from Panreac (Spain).
The mean size and SD were calculated by observing 200 ZnO-NPs in random view fields. The particle size distribution appeared to be approximately log-normal with 75 % of the particles (by number) having diameters from 20 to 80 nm. The mean ± SD of the NPs was 58.40 ± 30.13 nm.

Vehicle:

no

Details on preparation and application of test substrate:

The soil for ecotoxicity testing was collected from the top soil layer (0- to 20-cm soil depth excluding the vegetal cover) of a field located near Madrid (Spain)

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

Eisenia fetida (Oligochaeta:Lumbricidae) were obtained from own laboratory cultures. Clitellated adults (300 to 500 mg/individual) were kept on moist filter paper for 24 h to void the contents of their guts; then they were washed, dried, and weighed before being placed in test units

Study type:

laboratory study

Substrate type:

natural soil

Limit test:

yes

Total exposure duration:

28 d

Test temperature:

20+/-2°C

pH:

6.8

Moisture:

50% WTC (water holding capacity)

Details on test conditions:

The main physicochemical characteristics of this soil were as follows: clay 7.8 %; silt 18.8 %; sand 73.4 % (pH 6.8), and organic matter (OM) 1.9 %.

Nominal and measured concentrations:

nominal 1000mgZn/kg dw

Reference substance (positive control):

no

Duration:

24 d

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

element (total fraction)

Remarks:

from nano ZnO

Basis for effect:

mortality

Duration:

24 d

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

element (total fraction)

Remarks:

from ZnCl2

Basis for effect:

mortality

Duration:

24 d

Dose descriptor:

other: cocoons/worms/month

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

element (total fraction)

Remarks:

from nano ZnO

Basis for effect:

other: fecundity

Remarks on result:

other: 1.08+/-0.32 cocoons

Duration:

24 d

Dose descriptor:

other: cocoons/worms/month

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

element (total fraction)

Remarks:

from ZnCL2

Basis for effect:

other: fecundity

Remarks on result:

other: 0.03+/-0.05 cocoons

Reported statistics and error estimates:

The data were analyzed statistically using STATGRAPHICS software (version 5.0). Statistically significant differences between individual means for chemical and toxicological data were identify by analysis of variance with Fisher’s least significant difference procedure (LSD; p\0.05).

Validity criteria fulfilled:

yes

Conclusions:

No mortality was detected in any of Zn treatments or the control after 28 days. Fecundity was lower for ZnCl2 than for ZnO NP.

Reference 12

Endpoint:

toxicity to soil macroorganisms except arthropods: long-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: no guideline followed, no comparison to bulk or soluble zinc compound

Qualifier:

no guideline followed

Principles of method if other than guideline:

Adult E. fetida (with developed clitella) were exposed to varying concentrations of nano-sized ZnO in sand: 0, 0.1, 1, 10 and 1000 mg /kg. At the end of each exposure period, the number of cocoons produced, number of alive and dead adult worms, and worm weights were recorded.

GLP compliance:

no

Specific details on test material used for the study:

Zinc oxide nanoparticles (nanopowder, 40-100 nm particle size) were purchased from Alfa Aesar (Ward Hill, Maryland).

Analytical monitoring:

yes

Vehicle:

no

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Study type:

laboratory study

Substrate type:

other:

Limit test:

no

Total exposure duration:

21 d

Reference substance (positive control):

no

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

> 10 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

ZnO

Basis for effect:

reproduction

Remarks:

cocoon

Details on results:

Cocoon production was highest in the control group in the sandmanure test with nano-sized ZnO (n = 2) . As observed in the artificial soil, cocoon production decreased with an increase in metal oxide concentration.

DLS data indicate that for ZnO particles, the Z-average diameter shows a considerable increase as concentration is increased from 1 mg L1 to 100 mg L1, indicating aggregation.

Validity criteria fulfilled:

no

Reference 13

Endpoint:

toxicity to soil macroorganisms except arthropods: long-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: no guideline followed, no comparison to bulk or soluble zinc compound

Qualifier:

no guideline followed

Principles of method if other than guideline:

Adult E. fetida (with developed clitella) were exposed to varying concentrations of nano-sized ZnO in artificial soil: 0, 0.1, 1, 10 and 1000 mg/ kg. At the end of each exposure period, the number of cocoons produced, number of alive and dead adult worms, and worm weights were recorded.

GLP compliance:

no

Specific details on test material used for the study:

Zinc oxide nanoparticles (nanopowder, 40-100 nm particle size) were purchased from Alfa Aesar (Ward Hill, Maryland).

Analytical monitoring:

yes

Vehicle:

no

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

21 d

Reference substance (positive control):

no

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

ZnO

Basis for effect:

reproduction

Remarks:

cocoon

Details on results:

Cocoon production was highest in the control group in the artificial soil test with nano-sized ZnO. Cocoon production decreased with an increase in ZnO concentration. Number of cocoons decreased from three (at 0.1 mg kg1) to two (at 100 mg kg1) to 0 at the highest concentration (1000 mg kg1) of nano-sized ZnO. Although there was a slight increase in cocoon production at 10 mg kg1 (n=5), this value did not influence the trend of decreasing cocoon production. There was a significant difference between the control and 1000 mg kg1 exposure group (p = 0.0341). There was also a significant dose-response between ZnO concentration and cocoon production (p=0.0143).

DLS data indicate that for ZnO particles, the Z-average diameter shows a considerable increase as concentration is increased from 1 mg L1 to 100 mg L1, indicating aggregation.

When E. fetida were exposed to nano-sized ZnO in artificial soil, cocoon production significantly decreased in a dose dependent manner. Observed toxicity may be explained by the solubility of ZnO which could result in dissociation into free Zn2+ ions.

Validity criteria fulfilled:

no

Reference 14

Endpoint:

toxicity to soil macroorganisms except arthropods: 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: no guideline followed, no comparison to bulk or soluble zinc compound

Qualifier:

no guideline followed

Principles of method if other than guideline:

Adult E. fetida, identified by distinguishable clitella, were exposed to varying concentrations of either nano-sized ZnO on filter paper: 0, 0.1, 1, 10, 100, 1000, 5000 and 10 000 mg/L.

GLP compliance:

no

Specific details on test material used for the study:

Zinc oxide nanoparticles (nanopowder, 40100 nm particle size) were purchased from Alfa Aesar (Ward Hill, Maryland).

Analytical monitoring:

yes

Vehicle:

no

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Study type:

laboratory study

Substrate type:

filter paper

Limit test:

no

Total exposure duration:

14 d

Reference substance (positive control):

no

Duration:

14 d

Dose descriptor:

NOEC

Effect conc.:

> 10 000

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

ZnO

Basis for effect:

mortality

Remarks on result:

other: acute filter paper test

Details on results:

When exposed to nano-sized ZnO on filter paper, mortality in all groups except the control was high. There was no mortality in the controls; however, earthworm mortality did not exhibit a clear dose response (p = 0.098).

DLS data indicate that for ZnO particles, the Z-average diameter shows a considerable increase as concentration is increased from 1 mg L1 to 100 mg L1, indicating aggregation.

On filter paper, nano-sized ZnO did not significantly demonstrate acute toxicity (p > 0.05); however, mortality was high in all exposure groups with no survival at the highest concentration (10 000 mg L1).

Validity criteria fulfilled:

no

Reference 15

Endpoint:

toxicity to soil macroorganisms except arthropods: 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: no guideline followed, no comparison to bulk or soluble zinc compound

Qualifier:

no guideline followed

Principles of method if other than guideline:

Adult E. fetida, identified by distinguishable clitella, were exposed to varying concentrations of either nano-sized ZnO on sand: 0, 0.1, 1, 10, 100, 1000, 5000 and 10 000 mg/L.

GLP compliance:

no

Specific details on test material used for the study:

Zinc oxide nanoparticles (nanopowder, 40100 nm particle size) were purchased from Alfa Aesar (Ward Hill, Maryland).

Analytical monitoring:

yes

Vehicle:

no

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Study type:

laboratory study

Substrate type:

other: sand

Limit test:

no

Total exposure duration:

14 d

Reference substance (positive control):

no

Duration:

14 d

Dose descriptor:

NOEC

Effect conc.:

> 10 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

ZnO

Basis for effect:

mortality

Details on results:

Following exposure to nano-sized ZnO, E. fetida survived at every concentration with no mortality in the controls, 0.1, 1, 1000
and 5000 mg kg1 exposure groups. Earthworm mortality was ten percent at 10 and 100 mg kg1 and twenty percent at 10,000 mg kg1 when exposed to nano-sized ZnO. There was no significant dose-response between ZnO concentration and mortality of earthworms

DLS data indicate that for ZnO particles, the Z-average diameter shows a considerable increase as concentration is increased from 1 mg L1 to 100 mg L1, indicating aggregation.

Following exposure to nano-sized ZnO, E. fetida survived at every concentration with no mortality in the controls, 0.1, 1, 1000 and 5000 mg kg1 exposure groups. Earthworm mortality was ten percent at 10 and 100 mg kg1 and twenty percent at 10,000 mg kg1 when exposed to nano-sized ZnO. There was no significant dose-response between ZnO concentration and mortality of earthworms

Validity criteria fulfilled:

no

Reference 16

Endpoint:

toxicity to soil macroorganisms except arthropods: long-term

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 222 (Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei))

Principles of method if other than guideline:

Earthworm tests were performed using soils incubated for 1 d, 56 d, and 140 d after spiking.

GLP compliance:

not specified

Remarks:

GLP compliance not specified in paper

Specific details on test material used for the study:

ZnO-NPs (Nanosun Zinc Oxide P99/30) with a primary particle diameter size of 20 nm to 40 nm

Analytical monitoring:

yes

Details on sampling:

To check spiked concentrations, approximately 0.1 g ovendried soil samples were digested in 2mL of a 4:1 mixture of nitric acid (65% pro analysis; Riedel-de Haen) and hydrochloric acid (37% pro analysis; Baker) in tightly closed Teflon containers, which were heated in an oven at 140 °C for 7 h. Measured Zn concentrations were used in all data analyses. To determine the Zn concentration in earthworms, 1 freeze-dried individual earthworm of each replicate test container was digested using the same acid mixture and procedure as described for soil samples (n=4).

Vehicle:

no

Details on preparation and application of test substrate:

Three uncontaminated soils with contrasting properties were selected from different countries. Two soils were collected from the surface horizon of fields in Spain (SPCA; forestland in Granada) and The Netherlands (NLGA; a garden in Bilthoven), homogenized, 5mm–sieved, and air-dried. The third soil was the LUFA 2.2 natural standard soil (LUFA Speyer).

Test organisms (species):

Eisenia andrei

Animal group:

annelids

Details on test organisms:

Earthworms (E. andrei) were obtained from a laboratory culture at the Department of Ecological Science, Vrije Universiteit, Amsterdam, The Netherlands. Earthworms were fed with horse manure free of pharmaceuticals and incubated at 20 °C. The tests used adult earthworms with a well-developed clitellum, which were acclimatized for 24 h in the respective control soils before starting the exposures.

Study type:

laboratory study

Substrate type:

natural soil

Limit test:

yes

Total exposure duration:

56 d

Remarks:

28-d exposure of adult animals followed by another 28-d incubation of cocoons to enable the assessment of juvenile production.

Test temperature:

20°C

pH:

pH 5.6-7.6

Moisture:

Water holding capacity 15-62 %

Details on test conditions:

The ZnO-NPs were mixed into the soils as a powder to avoid dissolution of the particles prior to addition to the soil, while ZnCl2 was introduced as an aqueous solution. Soils were intensively mixed with a spoon to avoid modifying the NPs, to achieve as homogenous a distribution of the Zn as possible. After spiking, soils were moistened to 50% of their water holding capacity. Soils were dosed as a single batch, which was then split into separate aliquots for each time point and replicate. Soils were incubated in a climate-controlled room (Weiss Technik Benelux) at 20 8C and 75% relative air humidity, with a 12:12-h light: dark cycle. Soil moisture content was checked weekly by weighing the test containers and, if needed, readjusted.

Nominal and measured concentrations:

The selected dosing levels were adjusted to take into account the influence of soil pH and CEC on the effect concentrations. Test concentrations of ZnCl2 were 500 mg Zn kg-1 in LUFA and NLGA and 1250 mg Zn kg-1 in SPCA; ZnO-NP concentrations were 500 mg Zn kg-1 and 1000 mg Zn kg-1 in LUFA and NLGA and 1250 mg Zn kg-1 and 2500 mg Zn kg-1 in SPCA. Uncontaminated controls were also included. Measured concentrations (mean+/-SD) in the test soils on average were 97 +/- 6% (n=48) of the nominal (background and added) Zn concentrations, and variation among replicate samples was <18.5% for all treatments

Reference substance (positive control):

no

Duration:

56 d

Dose descriptor:

other: effect concentration (single [dose])

Effect conc.:

500 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

element (total fraction)

Remarks:

ZnCl2

Basis for effect:

reproduction

Remarks:

% reduction

Remarks on result:

other: 100%

Remarks:

LUFA soil, aged 1 day

Duration:

56 d

Dose descriptor:

other: effect concentration (single [dose])

Effect conc.:

500 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

element (total fraction)

Remarks:

ZnO nano

Basis for effect:

reproduction

Remarks:

% reduction

Remarks on result:

other: 31%

Remarks:

LUFA soil, aged 1 day

Duration:

56 d

Dose descriptor:

other: effect concentration (single [dose])

Effect conc.:

500 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

element (total fraction)

Remarks:

ZnCl2

Basis for effect:

reproduction

Remarks:

% reduction

Remarks on result:

other: 100%

Remarks:

LUFA soil, aged 56 days

Duration:

56 d

Dose descriptor:

other: effect concentration (single [dose])

Effect conc.:

500 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

element (total fraction)

Remarks:

ZnO nano

Basis for effect:

reproduction

Remarks:

% reduction

Remarks on result:

other: 89%

Remarks:

LUFA soil, aged 56 days

Duration:

56 d

Dose descriptor:

other: effect concentration (single [dose])

Effect conc.:

500 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

element (total fraction)

Remarks:

ZnCl2

Basis for effect:

reproduction

Remarks:

% reduction

Remarks on result:

other: 100%

Remarks:

LUFA soil, aged 140 days

Duration:

56 d

Dose descriptor:

other: effect concentration (single [dose])

Effect conc.:

500 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

element (total fraction)

Remarks:

ZnO nano

Basis for effect:

reproduction

Remarks:

% reduction

Remarks on result:

other: 100%

Remarks:

LUFA soil, aged 140 days

Duration:

56 d

Dose descriptor:

other: effect concentration (single [dose])

Effect conc.:

500 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

element (total fraction)

Remarks:

ZnCl2

Basis for effect:

reproduction

Remarks:

% reduction

Remarks on result:

other: 98%

Remarks:

NLGA soil, aged 1 day

Duration:

56 d

Dose descriptor:

other: effect concentration (single [dose])

Effect conc.:

500 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

element (total fraction)

Remarks:

ZnO nano

Basis for effect:

reproduction

Remarks:

% reduction

Remarks on result:

other: 41%

Remarks:

NLGA soil, aged 1 day

Duration:

56 d

Dose descriptor:

other: effect concentration (single [dose])

Effect conc.:

500 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

element (total fraction)

Remarks:

ZnCl2

Basis for effect:

reproduction

Remarks:

% reduction

Remarks on result:

other: 94%

Remarks:

NLGA soil, aged 56 days

Duration:

56 d

Dose descriptor:

other: effect concentration (single [dose])

Effect conc.:

500 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

element (total fraction)

Remarks:

ZnO nano

Basis for effect:

reproduction

Remarks:

% reduction

Remarks on result:

other: (-)5% (increased reprod.)

Remarks:

NLGA soil, aged 56 days

Duration:

56 d

Dose descriptor:

other: effect concentration (single [dose])

Effect conc.:

500 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

element (total fraction)

Remarks:

ZnCl2

Basis for effect:

reproduction

Remarks:

% reduction

Remarks on result:

other: 82%

Remarks:

NLGA soil, aged 140 days

Duration:

56 d

Dose descriptor:

other: effect concentration (single [dose])

Effect conc.:

500 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

element (total fraction)

Remarks:

ZnO nano

Basis for effect:

reproduction

Remarks:

% reduction

Remarks on result:

other: 22%

Remarks:

NLGA soil, aged 140 days

Duration:

56 d

Dose descriptor:

other: effect concentration (single [dose])

Effect conc.:

1 250 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

element (total fraction)

Remarks:

ZnCl2

Basis for effect:

reproduction

Remarks:

% reduction

Remarks on result:

other: 100%

Remarks:

SPCA soil, aged 1 day

Duration:

56 d

Dose descriptor:

other: effect concentration (single [dose])

Effect conc.:

1 250 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

element (total fraction)

Remarks:

ZnO nano

Basis for effect:

reproduction

Remarks:

% reduction

Remarks on result:

other: 45%

Remarks:

SPCA soil, aged 1 day

Duration:

56 d

Dose descriptor:

other: effect concentration (single [dose])

Effect conc.:

1 250 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

element (total fraction)

Remarks:

ZnCl2

Basis for effect:

reproduction

Remarks:

% reduction

Remarks on result:

other: 100%

Remarks:

SPCA soil, aged 56 days

Duration:

56 d

Dose descriptor:

other: effect concentration (single [dose])

Effect conc.:

1 250 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

element (total fraction)

Remarks:

ZnO nano

Basis for effect:

reproduction

Remarks:

% reduction

Remarks on result:

other: 41%

Remarks:

SPCA soil, aged 56 days

Duration:

56 d

Dose descriptor:

other: effect concentration (single [dose])

Effect conc.:

1 250 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

element (total fraction)

Remarks:

ZnCl2

Basis for effect:

reproduction

Remarks:

% reduction

Remarks on result:

other: 100%

Remarks:

SPCA soil, aged 140 days

Duration:

56 d

Dose descriptor:

other: effect concentration (single [dose])

Effect conc.:

1 250 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

element (total fraction)

Remarks:

ZnO nano

Basis for effect:

reproduction

Remarks:

% reduction

Remarks on result:

other: 75%

Remarks:

SPCA soil, aged 140 days

Details on results:

Survival was >78% for all treatments and sampling times, except for SPCA spiked with ZnCl2 after 56 d of aging where survival was only 25%. Earthworm weight loss, after 28 d of exposure, increased significantly with aging in all test soils (Tukey, p<0.05) except for NLGA spiked with ZnO-NPs (500 mg kg-1). The number of juveniles produced per earthworm during the 28-d exposure period in the controls was 1.8 in NLGA, 3.1 in LUFA, and 3.6 in SPCA for exposures starting after 1 d aging (data not shown). Compared to the control, earthworm reproduction was most affected by ZnCl2, with complete inhibition in LUFA and SPCA at all aging times and with >=82% reduction in NLGA. The LUFA soil also showed a significant decrease in earthworm reproduction after 140-d aging for both ZnO-NP treatments (Tukey, p<0.05). In NLGA, no significant reduction (p>0.05) in reproduction with time was seen for the ZnO-NP treatments. In SPCA, earthworm reproduction with time was not affected by the ZnO-NP treatment of 1250 mg Zn kg-1 soil, whereas at 2500 mg kg-1 almost no reduction was seen compared to the control after 56 d.

Reported statistics and error estimates:

Normal distribution of the data was verified using a Kolmogorov-Smirnov test. Significant differences were determined by analysis of variance, and multiple comparisons were performed with Tukey’s test (p<0.05). Partition coefficients (KdPW) were calculated as ZnT (milligrams per kilogram) divided by ZnPW (milligrams per kilogram). To compare soils and treatments with different Zn concentrations, bioaccumulation factors (BAFs) for the accumulation of Zn in the earthworms were calculated by dividing ZnE by ZnT. To determine the influence of soil properties and aging on Zn availability and earthworm responses for the 3 test soils, principal component analyses were performed using the CANOCO for Windows program, Ver 4.02. To study the effect of aging by relating soil properties and earthworm behavior, we assumed that soils, which were incubated under controlled conditions, had reached equilibrium well before day 140. The principal component analyses were done with the data of the earthworm exposures that started on day 140 and the chemical analysis data from day 168, the latter date coinciding with the end of the 28-d exposure of the earthworms. Ordination diagrams were explained with soils shown as points and most of the studied variables (KdPW, pHPW, BAF, ZnE, and weight loss).

Bioaccumulation factors calculated for the controls with the Zn background differed among the soils with the following pattern: LUFA>NLGA>SPCA. In the treatments with Zn, BAF was also lowest in the SPCA soil, whereas NLGA and LUFA had similar BAF values. The SPCA soil had the highest BAF in the lowest treatment with ZnO-NPs (1250 mg Zn kg-1 soil). At the same concentration added as ZnCl2, BAFs were similar to those in the ZnO-NP treatment of 2500 mg Zn kg-1 soil. It should be noted that earthworm survival was only 25% in SPCA soil spiked with ZnCl2 for exposures started after 56 d, making the BAF estimate less reliable. Except for the highest ZnO-NP treatment, BAF values in SPCA decreased with aging. In LUFA and NLGA, BAFs were higher in the ZnCl2 treatments and did not show clear trends with aging, whereas for the

Validity criteria fulfilled:

yes

Conclusions:

Although classic toxicity endpoints are not reported (EC or IC), still useful for determinig comparative effects on reproduction for ZnCl2 versus ZnO nano.

Executive summary:

Good study, well documented. For all 3 natural soils tested (LUFA, NLGA, SPCA), all aged for 3 differernt durations (1, 56 and 140 days), ion toxicity from ZnCl2 excerted a greater reduction in earthworm reproduction success.

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.

Taking into account the general observed trend, the PNEC derivation for soil, based on the soluble zinc ion, is considered relevant for the nano-ZnO form, too.

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 (attached background material) 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.

Taking into account the general observed trend, the PNEC derivation for soil, based on the soluble zinc ion, is considered relevant for the nano-ZnO form, too.