Zinc oxide

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to microorganisms

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Good quality study but not following guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

A pulse-flow PF-8000 aerobic/anaerobic respirometer (Respirometer Systems and Applications, Fayetteville, Arkansas, USA) was used to determine the effect of different Zn forms (nano-ZnO, bulk ZnO, and Zn ion) on activated sludge endogenous respiration, BOD biodegradation, and nitrification. The experimental system consisted of a pure oxygen supply unit, a control module, eight bioreactors in a water bath, and a computer. The biological activity in the bioreactor was monitored by measuring the oxygen uptake rate (OUR).

GLP compliance:

not specified

Specific details on test material used for the study:

Nano-ZnO (10–30 nm) was purchased from SkySpring Nanomaterials, Inc. (Houston, TX, USA). Bulk ZnO powder was from Fisher Scientific. Soluble Zn stock solution (5000 mg-Zn/L) was made of Zn (NO3)2·6H2O from Fisher Scientific.

Vehicle:

no

Test organisms (species):

activated sludge

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

10 h

Test temperature:

20°C

Details on test conditions:

The same activated sludge volume of 500 mL was used in all experiments. The mixed liquor suspended solid (MLSS) concentration and the mixed liquor volatile suspended solid (MLVSS) concentration were approximately 3.0 g/L and 2.3 g/L, respectively. In each batch of the experiment, all but one reactorswere addedwith different amounts of test chemicals (nano-ZnO, bulk ZnO, or soluble Zn). One reactor was used as a control, without adding any chemical. Weighed amounts of nano-ZnO and bulk ZnO were added to reactors as dry powder, while selected volumes of stock Zn solutionwere added using a pipette. Before adding chemicals, open reactor bottles were placed in a water bath at a controlled temperature of 20 °C and mixed for 20 min to equalize the temperature and dissolved oxygen. All reactors were then added with selected amounts of chemicals (except the control) and sealed. As soon as the reactor was sealed, the OUR was recorded.

Duration:

10 h

Dose descriptor:

IC50

Effect conc.:

13.1 mg/L

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

growth inhibition

Remarks on result:

other: nano ZnO

Duration:

10 h

Dose descriptor:

IC50

Effect conc.:

7.5 mg/L

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

growth inhibition

Remarks on result:

other: bulk ZnO

Duration:

10 h

Dose descriptor:

IC50

Effect conc.:

6.5 mg/L

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

growth inhibition

Remarks on result:

other: zinc salt

Validity criteria fulfilled:

yes

Executive summary:

In general, added soluble Zn was more toxic than added nano-ZnO, and added nano-ZnO was more toxic than added bulk ZnO, to all

biological reactions including endogenous respiration, BOD biodegradation, and both steps of nitrification. However, all inhibition effects of nano-ZnOand bulk ZnOwere derived from soluble Zn that resulted from particle dissolution. The IC50 values of soluble Zn for endogenous respiration, BOD biodegradation, the first step of nitrification (e.g., ammonia oxidation), and the second step of nitrification (e.g., nitrite oxidation) were 2.2, 1.3, 0.8, and 7.3 mg-Zn/L, respectively. Therefore, the first step of nitrification was more sensitive to Zn than other reactions. For a wastewater that contained 3 g/L activated sludge, most nano-ZnO and all bulk ZnO were dissolved when added particle concentration was below 16mg-Zn/L. Further increasing ZnO addition resulted in only partial dissolution of both nano-ZnO and bulk ZnO. The dissolution profiles were in agreement with the toxicity data for different forms of Zn.

Reference 2

Endpoint:

toxicity to microorganisms

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable, well documented publication/study report which meets basic scientific principles

Qualifier:

no guideline followed

Principles of method if other than guideline:

Lab specific dose-response experiment

GLP compliance:

not specified

Specific details on test material used for the study:

Nano-ZnO (advertised particle size 50–70 nm) was purchased fromSigma–Aldrich and analysed in parallel with bulk ZnO (Fluka). ZnSO4 ×7H2O served as ionic controls

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

The stock suspensions/solutions of the tested chemicals were prepared in deionised water (MilliQ, Millipore). The stock suspensions
of metal oxides (40 g/l) were sonicated for 30 min, stored in the dark at +4 ◦C and used for testing within 2 months. Stock solutions of ZnSO4 ×7H2O was prepared analogously, but not sonicated. Before toxicity testing, stocks were vigorously vortexed. The aqueous suspensions of the studied metal oxides (both nano- and bulk formulations) have been previously characterized by scanning electron microscopy (SEM): despite of agglomeration, individual nanoscale particles were present in nano-ZnO suspensions (Kahru et al., 2008). Osterhout’s medium was used throughout the experiments as a diluent and a control.

Test organisms (species):

Tetrahymena sp.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

24 h

Test temperature:

25°C

pH:

6.5

Nominal and measured concentrations:

2.84, 5.69, 11.37, 22.75, 45.49mg Zn2+/l of ZnSO4 ×7H2O;
1.85, 5.55, 8.33, 12.5, 25 mg/l of nano- and bulk ZnO.

Reference substance (positive control):

no

Duration:

4 h

Dose descriptor:

EC50

Effect conc.:

4.3 mg/L

Nominal / measured:

nominal

Conc. based on:

dissolved

Remarks:

Zinc

Basis for effect:

other: viability

Remarks on result:

other: nano ZnO fluorescence

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

6.8 mg/L

Nominal / measured:

nominal

Conc. based on:

dissolved

Remarks:

Zinc

Basis for effect:

other: viability

Remarks on result:

other: nano ZnO fluorescence

Duration:

4 h

Dose descriptor:

EC50

Effect conc.:

3.9 mg/L

Nominal / measured:

nominal

Conc. based on:

dissolved

Remarks:

ZInc

Basis for effect:

other: viability

Remarks on result:

other: bulk ZnO fluorescence

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

7.4 mg/L

Nominal / measured:

nominal

Conc. based on:

dissolved

Remarks:

Zinc

Basis for effect:

other: viability

Remarks on result:

other: ZnO bulk fluorescence

Duration:

4 h

Dose descriptor:

EC50

Effect conc.:

4.5 mg/L

Nominal / measured:

nominal

Conc. based on:

dissolved

Remarks:

Zinc

Basis for effect:

other: viability

Remarks on result:

other: ZnSO4 fluorescence

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

6.7 mg/L

Nominal / measured:

nominal

Conc. based on:

dissolved

Remarks:

Zinc

Basis for effect:

other: viability

Remarks on result:

other: ZnSO4 fluorescence

Reported statistics and error estimates:

ANOVA p<0.05

Validity criteria fulfilled:

yes

Executive summary:

One may think that particle-feeding organism should be more susceptible to toxic effects of particulate compounds than organisms not internalising particles. On the contrary,this study showed that, even though intensively accumulated in the food vacuoles, the toxicity of nanoparticles of ZnO to T. thermophila was due to dissolved metal ions, analogously to bacteria, algae and crustaceans.

Reference 3

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Name of test material (as cited in study report):NM-110 Zinc Oxide
- Physical state: Solid/ White
- Analytical purity: 99.1g/100 g
- Lot/batch No.: NM-110 Refernce Nanomaterial

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Laboratory culture:Activated sludge from the municipal wastewater treatment plant of Mannheim, Germany was colledted from the aeration tank of the plant.
- Preparation of inoculum for exposure: The activated sludge suspension was sieved with a fine woven mesh (mesh size about 1 mm). This suspension was pre-aerated over night at room temperature. At the next day the sludge suspension was washed once with drinking water and the suspension was adjusted to 3 g/L dry matter.
- Pretreatment: none
- Initial biomass concentration: 1.5 g/L dry substance

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

180 min

Details on test conditions:

TEST SYSTEM
- Test vessel: Glas-beakers (nominal volume 1L)
- Type (delete if not applicable): closed
- Aeration: the incubation was started by aeration of the test vessels with pressured air
- Type of flow-through (e.g. peristaltic or proportional diluter):
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): 2 for each reference substance concentration

OTHER TEST CONDITIONS
- Adjustment of pH: no

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Justification for using less concentrations than requested by guideline:
- Test concentrations: 1000, 500, 250, 125, 62,5 mg/l

Reference substance (positive control):

yes

Remarks:

3,5 dichlorophenol

Duration:

180 min

Dose descriptor:

EC10

Effect conc.:

720 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: 391.8/1319.1

Duration:

180 min

Dose descriptor:

other: EC20

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Duration:

180 min

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Results with reference substance (positive control):

The EC50 of the reference substance 3,5-dichlorophenol was in the range of 2-25 mg/L in 3 hours.

The value of effect concentration of EC10 was given with an accuracy of 2 significant digits. The degree of inhibition was evaluated by Probit analysis according to Finney.

The results in this study were consistent with the validity criteria with one exception. The mean oxygen uptake in the blank controls was lower than 20 mg/g*h (17 mg/g*h). Because the reference substance shows an EC50 in the specified range (usual range of EC50 in the

laboratory was 4.5 – 11.9 mg/L in the year 2012) and the measured oxygen uptake from the test substance concentrations showed a good curve progression the study is classified as valid. In the year 2012 the used activated sludge which was collected from the same waste water treatment plant showed a maximum oxygen consumption of 35.8 mg/g*h and minimum oxygen consumption of 10.5 mg/g*h.

Description of key information

Four studies were done to assess the toxicity of zinc oxide nanoforms on microorganisms. A study by Blinova et al (2010) on the ecotoxicity of CuO and ZnO nanoparticles in natural water, a study by Mortimer et al (2010) on the toxicity of ZnO and CuO nanoparticles to ciliated protozoa Tetrahymena thermophila, a study by Liu et al (2011) on the effect of ZnO particles on activated sludge: role of particle dissolution and a study by BASF (2012) on the determination of Oxygen Consumption inhibition of ZnO NM-110 in the Activated Sludge Respiration Inhibition Test.

The studies by Blinova et al (2010) and Mortimer et al (2010) showed that, even though intensively accumulated in the food vacuoles, the toxicity of nanoparticles of ZnO to T. thermophila was due to dissolved metal ions, analogously to bacteria, algae and crustaceans.

The study by Liu et al (2011) showed that added soluble Zn was more toxic than added nano-ZnO, and added nano-ZnO was more toxic than added bulk ZnO, to all biological reactions including endogenous respiration, BOD biodegradation, and both steps of nitrification. However, all inhibition effects of nano-ZnO and bulk ZnO were derived from soluble Zn that resulted from particle dissolution.

The study by study by BASF (2012) measured an EC50 for the inhibition of total respiration (respiration rate) of activated sludge from a municipal wastewater treatment plant of > 1000 mg/L for test item ZnO NM-110.

Additional information

The study by Blinova et al (2010) was done with two forms of ZnO: "bulk" ZnO (Fluka), and "nano"-ZnO, with particle size 70nm from Sigma-Aldrich. The substance was added to the food substrate suspension in the test medium. Zn background of natural waters varied between 1.4 and 3.1 mg (total)/l. This study showed that, even though intensively accumulated in the food vacuoles, the toxicity of nanoparticles of ZnO to T. thermophila was due to dissolved metal ions, analogously to bacteria, algae and crustaceans.

The study by Mortimer et al (2010) was done with stock suspensions/solutions of Nano-ZnO (particle size 50–70 nm) analysed in parallel with bulk ZnO (Fluka). ZnSO4 ×7H2O served as ionic controls prepared in deionised water (MilliQ, Millipore). The test was done at 25°C for a total exposure duration of 24 at pH 6.5 and concentrations of 2.84, 5.69, 11.37, 22.75, 45.49mg Zn2+/l of ZnSO4 ×7H2O and concentrations of 1.85, 5.55, 8.33, 12.5, 25 mg/l of nano- and bulk ZnO. Contrary to what may seem (particle-feeding organism could be more susceptible to toxic effects of particulate compounds than organisms not internalising particles), this study showed that even though intensively accumulated in the food vacuoles, the toxicity of nanoparticles of ZnO to T. thermophila was due to dissolved metal ions, analogously to bacteria, algae and crustaceans.

The study by Liu et al (2011) was done with Nano-ZnO (10–30 nm) and Bulk ZnO powder. A pulse-flow PF-8000 aerobic/anaerobic respirometer (Respirometer Systems and Applications, Fayetteville, Arkansas, USA) was used to determine the effect of different Zn forms (nano-ZnO, bulk ZnO, and Zn ion) on activated sludge endogenous respiration, BOD biodegradation, and nitrification.
In general, added soluble Zn was more toxic than added nano-ZnO, and added nano-ZnO was more toxic than added bulk ZnO, to all biological reactions including endogenous respiration, BOD biodegradation, and both steps of nitrification. However, all inhibition effects of nano-ZnO and bulk ZnO were derived from soluble Zn that resulted from particle dissolution. For a wastewater that contained 3 g/L activated sludge, most nano-ZnO and all bulk ZnO were dissolved when added particle concentration was below 16mg-Zn/L. Further increasing ZnO addition resulted in only partial dissolution of both nano-ZnO and bulk ZnO. The dissolution profiles were in agreement with the toxicity data for different forms of Zn.

The study by BASF (2012) was done on ZnO reference NM-110 according to OECD guideline 209. The concentrations of the test substance were 1000, 500, 250, 125 and 62,5 mg/l and the total exposure duration was 180min. The test was done on activated sludge from the municipal wastewater treatment plant of Mannheim, Germany, collected from the aeration tank of the plant. The activated sludge suspension was then sieved with a fine woven mesh and pre-aerated over night at room temperature. At the next day the sludge suspension was washed once with drinking water and the suspension was adjusted to 3 g/L dry matter. Its initial biomass concentration was 1.5 g/L dry substance. The EC50 for the inhibition of total respiration (respiration rate) was > 1000 mg/L.