lambda4-cerium(4+) zirconium(4+) tetraoxidandiide

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 14-DEC-2007 to 15-MAY-2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Triplicate samples were taken from the test medium and from the control just before the start of the test and after 24, 48 and 72 hours. The concentrations of cerium were analytically determined in two samples of the test media with the loading rates 3.2, 10, 32 and 100 mg/L and in two control samples taken at the start of the test and after 24, 48 and 72 hours. The samples from the lower test concentrations (loading rates 0.32 and 1.0 mg/L) were not analysed, since these test concentrations were far below the 72-hour NOELR and, thus, not relevant for the interpretation of the biological results.
- Sampling method: no data
- Sample storage conditions before analysis: Immediately after sampling, the samples were acidified with 10% (v/v) nitric acid to stabilise the test item during the storage period. Then the samples were stored in PE flasks at ambient temperature and protected from light until analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method:
The test item is a multi-constituent substance containing different sparingly soluble components. In order to assess its toxicity, a water accommodated fraction (WAF) was prepared. The test method was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures.
The following loading rates were tested: 0.32, 1.0, 3.2, 10, 32 and 100 mg/L. Additionally, a control was tested in parallel (test water without test item).
For preparation of the WAFs, individual dispersions of the test item with the loading rates of 3.2, 10, 32 and 100 mg/L were prepared using ultrasonic treatment for 15 minutes and intense stirring. The dispersions were stirred for 6 days to dissolve a maximum amount of the different compounds of the test item in the dispersion. The dispersions were stirred on magnetic stirrers at room temperature in the dark. After the stirring period, the stirrers were switched off to allow the non-dissolved test item to deposit at the bottom of the stirring vessels for another 24 hours. The total contact time of the test item and the test water for equilibration was 7 days. The WAFs with the two lowest loading rates of 0.32 and 1.0 mg/L were prepared by dilution of the WAF with the loading rate of 3.2 mg/L due to technical reasons.
The equilibrated test media were carefully separated from the non-dissolved test item and used as WAFs. The test media were prepared just before the start of the test (= addition of algae).
- Eluate: no
- Differential loading: yes
- Controls: blank (test water without test item)
- Evidence of undissolved material (e.g. precipitate, surface film, etc): yes, on the bottom of the stirring vessel, but not in the final test solution

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Scenedesmus subspicatus CHODAT
- Strain: No. 86.81 SAG
- Source: supplied by the Collection of Algal Cultures (SAG, Institut for Plant Physiology, University of Göttingen, 37073 Göttingen, Germany)
- Age of inoculum (at test initiation): The algal cells were taken from an exponentially growing pre-culture, which was set up four days prior to the test under the same conditions as in the test. One day before the start of the test, the pre-culture was diluted threefold to keep the algae in exponential growth.
- Method of cultivation: Cultivated in Harlan laboratories in synthetic test water, prepared according to the test guidelines. Analytical grade salts were dissolved in sterile purified water.

ACCLIMATION
- Acclimation period: four days
- Culturing media and conditions: see method of cultivation above
- Any deformed or abnormal cells observed: data not available

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

0.24 mmol/L (= 24 mg/L as CaCO3)

Test temperature:

22°C

pH:

8.1 - 8.5

Dissolved oxygen:

not measured

Salinity:

not applicable

Nominal and measured concentrations:

Nominal loading rates: 0.32 mg/L, 1.0 mg/L, 3.2 mg/L, 10 mg/L, 32 mg/L and 100 mg/L (= saturated solution)
Measured concentrations: 3 µg Ce/L or 6 µg test item/L (at 3.2 mg/L); <=1 µg Ce/L or <=2 µg test item/L (at 10 mg/L); 2 µg Ce/L or 4 µg test item/L (at 32 mg/L); 20 µg Ce/L or 42 µg test item/L (at 100 mg/L)

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type: Erlenmeyer flasks covered with glass dishes
- Material, size, headspace, fill volume: 50-mL flasks, filled with 15 mL of algal suspension
- Aeration: no
- Type of flow-through (e.g. peristaltic or proportional diluter): none (static test)
- Renewal rate of test solution (frequency/flow rate): a static, non-renewal exposure system was used.
- Initial cells density: 5000 algal cells per mL of test medium
- Control end cells density: 659 900, 99 algal cells per mL
- No. of vessels per concentration (replicates): three replicates
- No. of vessels per control (replicates): six replicates

GROWTH MEDIUM
- Standard medium used: The algae were cultivated in synthetic test water, prepared according to the test guidelines. Analytical grade salts were dissolved in sterile purified water.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: sterile purified water
- Total organic carbon, Particulate matter, Metals, Pesticides, Chlorine, Alkalinity, Ca/mg ratio, Conductivity: data not available
- Culture medium different from test medium: no
- Intervals of water quality measurement: The pH was measured and recorded in each test concentration and the control at the start and at the end of the test. The water temperature was measured and recorded daily in an Erlenmeyer flask filled with water and incubated under the same conditions as the test flasks. The appearance of the test media was also recorded daily. The concentration of phosphate was determined in duplicate in the test media and the control at the start of the test and then daily until the end of the test using a photometric method (Merck Spectroquant phosphate test 1.14848.0001). Prior to the determination, the algal cells were removed by filtration trough glass fibre microfilters (GF/C Whatman). The 24-hour, 48-hour and 72-hour samples were taken from the separately incubated test media with algae which were also used for analytical purposes.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuous illumination
- Light intensity and quality: The measured light intensity was about 7100 Lux (mean value) and was achieved by fluorescent tubes (Philips TLD 36W/840) installed above the test flasks.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Determination of cell concentrations: A small volume of the algal suspension was daily withdrawn from each test flask for the measurement of the biomass, and was not replaced. The algal biomass in the samples was determined by fluorescence measurement (BIOTEK® Multi-Detection Microplate Reader, Model FLx800). The measurements were performed at least in duplicate. Inhibition of algal growth was determined from: (i) the area under the growth curves (AUC), biomass integral, (ii) the specific growth rates (µ), and (iii) the yield (Y).
- Other: In addition, after 72 hours of exposure, a sample was taken from the control and from the WAF. The shape and size of the algal cells were examined microscopically in these samples.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Justification for using less concentrations than requested by guideline: not applicable
- Range finding study: yes
- Test concentrations of the range finding study: no data
- Results used to determine the conditions for the definitive study: An enlarged spacing factor of 3.2 between the test concentrations was chosen because, according to the results of the range-finding test, the concentration-effect relationship was rather flat and thus, a large concentration had to be tested.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI not determined

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 42 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Remarks:

calculated based on dissolved Ce monitoring

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

4 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Remarks:

calculated based on dissolved Ce monitoring

Basis for effect:

growth rate

Details on results:

BIOLOGICAL RESULTS
- Exponential growth in the control (for algal test): yes (in the control, the biomass increased by a factor of 132 over 72 hours)
- Observation of abnormalities (for algal test): no
- Other: The size and shape of the algal cells was not affected.

APPEARANCE OF THE TEST MEDIUM
No remarkable observations were made concerning the appearance of test medium. The test medium was a clear solution throughout the test period.

PHOSPHATE CONCENTRATIONS
The concentration of phosphate was statistically significantly reduced compared to the control in the WAFs with the loading rate of 32 mg/L and above (results of a Student-t test with Bonferroni correction, p<0.008). The loss of phosphate can be explained by the formation of insoluble complexes of phosphate with the test item (which is a well-known behaviour of rare earth elements in the environment). The depletion of phosphate in the test medium during the test might have been the reason for the inhibition of algal growth determined at this test concentration. Thus, growth inhibition due to a secondary effect (i.e. the complexation of the essential algal nutrient phosphate by the test item) cannot be excluded.

ANALYTICAL MONITORING
The concentrations of cerium were measured in samples taken daily from the WAFs with loading rates of 3.2, 10, 32 and 100 mg/L. The measured concentrations of cerium during the test were between 1 (limit of quantification) and 3 µg/L at the loading rates of 3.2 to 32 mg/L. At the highest loading rate of 100 mg/L, the concentrations of cerium were 27, 29, 19 and 4 µg/L at the start of the test and after 24, 48 and 72 hours, respectively.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- 72-hr EC50 for the growth rate = 0.64 mg/L (acceptance range: 0.44-1.16 mg/L) (potassium dichromate)

Reported statistics and error estimates:

The EC10 and EC50 values (the respective loading rates of the test item corresponding to 10 and 50% inhibition, respectively, compared to the control) for the different growth parameters and their 95% confidence intervals were calculated as far as possible by Probit Analysis. The EC90 could not be calculated for the different growth parameters because the inhibition of the parameters was far below 90% at all test concentrations.
For the determination of the LOEC and NOEC, the calculated AUC, the growth rate and the yield at the test concentrations were compared to the corresponding control values by multiple Dunnett's tests (one-sided, alpha = 0.05).

Table 1: Biomass of Algae

Treatment / Loading rate (mg/L)	Rep. no.	Biomass of algae* (relative Fluorescence units)
		24 hours	48 hours	72 hours
Control	1 2 3 4 5 6	6.6 6.7 7.1 6.6 6.5 6.5	34.1 36.8 39.5 34.5 30.1 35.2	130.8 136.5 140.5 139.7 116.6 135.5
	Mean SD	6.7 0.2	35.0 3.1	133.3 8.9
0.32	1 2 3	6.6 6.9 7.2	36.8 34.2 37.1	132.8 145.7 148.0
	Mean SD	6.9 0.3	36.0 1.6	142.2 8.2
1.0	1 2 3	6.5 6.7 6.2	33.1 35.2 32.8	145.9 145.8 145.5
	Mean SD	6.4 0.3	33.7 1.3	145.7 0.2
3.2	1 2 3	6.1 6.7 7.2	30.8 36.2 34.8	138.2 138.5 139.0
	Mean SD	6.7 0.5	33.9 2.8	138.6 0.4
10	1 2 3	6.6 6.0 6.0	30.9 32.9 34.2	151.4 146.0 151.4
	Mean SD	6.2 0.4	32.7 1.7	149.6 3.1
32	1 2 3	6.2 6.5 6.9	36.0 34.6 35.4	148.3 157.6 159.3
	Mean SD	6.5 0.4	35.3 0.7	155.1 5.9
100	1 2 3	6.3 6.9 6.9	26.6 23.9 26.8	46.7 48.9 45.7
	Mean SD	6.7 0.3	25.8 1.6	47.1 1.6

 

SD: Standard deviation

*: The biomass was determined by fluorescence measurement (duplicate measurements) and is given as relative fluorescence units (x 10 exp 3). At the start of the test, the initial cell density was 5000 algal cells/mL, corresponding to 1.01x 10 exp 3 relative fluorescence units).

 

Table 2: Areas under the Growth Curves (AUC)

Loading rate (mg/L)	Areas under the growth curves AUC (10 exp3 *day) And inhibition of AUC (IAUC)
	0-24 h		0-48 h		0-72 h
	AUC	IAUC(%)	AUC	IAUC(%)	AUC	IAUC(%)
Control	2.8	0.0	22.7	0.0	105.8	0.0
0.32	2.9	-3.5	23.4	-3.0	111.4	-5.3
1.0	2.7	3.8	21.8	3.9	110.5	-4.4
3.2	2.8	-0.1	22.1	2.4	107.4	-1.5
10	2.6	7.4	21.1	7.1	111.2	-5.1
32	2.8	2.5	22.7	0.0	116.8	-10.4
100	2.8	-0.6	18.1*	20.3	53.5*	49.4

*: mean value significantly lower than in the control

(according to Dunnett's tests, one-sided, alpha = 0.05)

 

Table 3: : Average Growth Rates (µ)

Loading rate (mg/L)	Average growth rate (day-1) and inhibition of (Ir)
	0-24 h		0-48 h		0-72 h
	µ	Ir (%)	µ	Ir (%)	µ	Ir (%)
Control	1.89	0.0	1.77	0.0	1.63	0.0
0.32	1.92	-1.5	1.79	-0.9	1.65	-1.3
1.0	1.86	1.8	1.76	1.0	1.66	-1.9
3.2	1.89	0.1	1.76	0.9	1.64	-0.8
10	1.82	3.5	1.74	1.9	1.67	-2.4
32	1.87	1.2	1.78	-0.3	1.68	-3.1
100	1.89	-0.2	1.62*	8.6	1.28*	21.3

*: mean value significantly lower than in the control

(according to Dunnett's tests, one-sided, alpha = 0.05)

 

Table 4: Yield (Y)

Loading rate (mg/L)	Yield Y and inhibition of Y (Iy)
	0-24 h		0-48 h		0-72 h
	Y	Iy (%)	Y	Iy (%)	Y	Iy (%)
Control	5.7	0.0	34.0	0.0	132.2	0.0
0.32	5.9	-3.5	35.0	-2.9	141.2	-6.7
1.0	5.4	3.8	32.7	3.9	144.7	-9.4
3.2	5.7	-0.1	32.9	3.3	137.6	-4.0
10	5.2	7.4	31.7	7.0	148.6	-12.4
32	5.5	2.5	34.3	-0.9	154.1	-16.5
100	5.7	-0.6	24.8*	27.2	46.1*	65.2

 

*: mean value significantly lower than in the control

(according to Dunnett's tests, one-sided, alpha = 0.05)

 

Table 5: Section-by-section growth rates

Loading rate (mg/L)	Section-by-section growth rates (day-1) and inhibition of the growth rates (Ir)
	0-24 h		24-48 h		48-72 h
	µ	Ir (%)	µ	Ir (%)	µ	Ir (%)
Control	1.89	0.0	1.66	0.0	1.34	0.0
0.32	1.92	-1.5	1.66	-0.1	1.37	-2.6
1.0	1.86	1.8	1.65	0.1	1.46	-9.5
3.2	1.89	0.1	1.63	1.8	1.41	-5.4
10	1.82	3.5	1.66	0.1	1.52	-13.8
32	1.87	1.2	1.69	-2.0	1.48	-10.6
100	1.89	-0.2	1.35	18.6	0.60	54.9

 

 Table 6: Phosphate concentrations in the test media and in the control

Loading rate (mg/L)	Phosphate (mg PO4/L)
	0 h			24 h			48 h			72 h
	Sample 1+ 2		mean	Sample 1+ 2		mean	Sample 1+ 2		mean	Sample 1+ 2		mean
Control	1.16	1.08	1.12	1.06	1.04	1.05	0.68	0.65	0.67	< 0.03	< 0.03	< 0.03
0.32	1.15	1.13	1.14	1.06	1.08	1.07	0.83	0.70	0.77	< 0.03	< 0.03	< 0.03
1.0	1.08	1.10	1.09	1.03	1.07	1.05	0.67	0.69	0.68	< 0.03	< 0.03	< 0.03
3.2	1.04	1.06	1.05	0.99	1.00	0.99	0.69	0.69	0.69	< 0.03	< 0.03	< 0.03
10	0.94	0.94	0.94	0.92	0.94	0.93	0.57	0.58	0.58	< 0.03	< 0.03	< 0.03
32	0.65	0.64	0.65	0.62	0.64	0.63	0.25	0.25	0.25	< 0.03	< 0.03	< 0.03
100	0.11	0.10	0.10	0.09	< 0.03	0.05	< 0.03	< 0.03	< 0.03	< 0.03	< 0.03	< 0.03

 

Validity criteria fulfilled:

yes

Remarks:

The control biomass is multiplicated by 132 over 72 hours (threshold > 16), the mean coeff. of variation of the daily growth rates was 17% (threshold < 35%), and the coeff. of variation of the average specific growth rates was 1.4% (threshold < 7%)

Conclusions:

The test item had a statistically significant inhibitory effect on the growth (based on AUC, growth rate and yield) of Scenedesmus subspicatus after the test period of 72 hours at the highest loading rate of 100 mg/L (measured concentration of 42 µg test item/L). Thus, this loading rate was determined as the 72-h LOEC. The 72-h EC50 was > 100 mg/L. The NOEC was determined to be 32 mg/L based on loading rate (measured: 4 µg test item/L). The loss of phosphate in the WAFs with the loading rate of 32 mg/L and above can be explained by the formation of insoluble complexes of phosphate with the test item (which is a well-known behaviour of rare earth elements as well as zirconium in the environment). The depletion of phosphate in the test medium during the test was clearly the reason for the inhibition of algal growth determined at this test concentration. Thus, growth inhibition was due to a secondary effect (i.e. the complexation of the essential algal nutrient phosphate by the test item). This secondary effect is not considered environmentally relevant.

Executive summary:

In a 72-hour toxicity study, cultures of the green algal species Scenedesmus subspicatus were exposed to the reaction mass of cerium dioxide and zirconium dioxide at the loading rates of 0.32, 1.0, 3.2, 10, 32 and 100 mg/L under static conditions in accordance with the EU Commission Directive 92/69/EEC, C.3 (1992), and OECD Guideline 201 (2006). The NOEC, LOEC and EC50 values based on growth rate were 32 mg/L, 100 mg/L and > 100 mg/L, respectively (based on nominal loading rates).   

 

This toxicity study is classified as acceptable and satisfies the guideline requirements for algal (Scenedesmus subspicatus) growth inhibition studies.

Additional remark:

The concentration of phosphate was statistically significantly reduced compared to the control in the WAFs with the loading rate of 32 mg/L and above (results of a Student-t test with Bonferroni correction, p<0.008). The loss of phosphate can be explained by the formation of insoluble complexes of phosphate with the test item (which is a well-known behaviour of rare earth elements as well as zirconium in the environment). The depletion of phosphate in the test medium during the test was clearly the reason for the inhibition of algal growth determined at this test concentration. Thus, growth inhibition was due to a secondary effect (i.e. the complexation of the essential algal nutrient phosphate by the test item) which is not considered environmentally relevant.

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Justification for type of information:

Read across from studies performed with micrometric and nanometric cerium dioxide. The read across justification document is attached in IUCLID Section 13.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Remarks on result:

other: Data obtained on micrometric and nanometric cerium dioxide are included in the dossier as supporting information.

Remarks:

In the case of studies carried out with micrometric forms of cerium dioxide, data concluded that cerium dioxide was not harmful to algae, either because no adverse effects had been observed up to and including at the highest concentration tested (Van Hoecke et al., 2009) or the observed adverse effects on growth were attributed to phosphate depletion from the test medium caused by complexation with the test item (Bätscher, 2007b). The available study on nanometric cerium dioxide (Van Hoecke et al., 2009) observed adverse effects from a concentration of 5.6 mg/L onwards. It could however not be excluded that clustering of the algal cells with the nanomaterial and/or phosphate depletion from the test medium were responsible for / contributed to the observed toxicity. Based on these results, and taking together all other information (e.g. further information from algal growth inhibition studies with zirconium substances, and from the study with a representative nanoform of the reaction mass), it is not expected that the reaction mass of cerium dioxide and zirconium dioxide would cause environmentally relevant harmful effects in algae.

Reference 3

Endpoint:

toxicity to aquatic algae and cyanobacteria

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

Remarks:

The study was conducted according to the OECD internationally recognised guideline, without reference to the application of GLP and without analytical monitoring of the test material. However, the experimental details and results were well described.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

not specified

Analytical monitoring:

no

Details on sampling:

No analytical monitoring of the test substance concentration during the test

Vehicle:

no

Details on test solutions:

- Method: Experimental test concentrations were prepared by dropwise addition of the cerium dioxide nanoparticles stock suspensions to the test medium adjusted to pH 4 using a 1 M HCl solution, while stirring. Subsequently, the pH of the test suspensions was adjusted to 7.4. Prior the pH adjustment, 750 mg/L MOPS (3-(N-morpholino)propanesulfonic acid) buffer was added to the media.
- Control: yes (test water without test item)
No further data

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: unicellular freshwater green alga
- Strain: Printz
- Source (laboratory, culture collection): The alga was obtained from the Culture Collection of Algae and Protozoa (CCAP 278/4, Oban, Scotland) and a culture was established at Ghent University (Belgium).
- Age of inoculum (at test initiation): no data
- Method of cultivation: The culture medium consisted of Es-medium at 1/2 strength which was added to carbon filtered aerated tap water (Ghent, Belgium), supplemented with 1.4 mg/L FeSO4.7H2O, 15 mg/L NaH2PO4.2H2O, 1510 mg/L NaNO3 and 2.35 mg/L MnCl4.4H2O. Each week, cultures were visually inspected for contamination using a light microscope. Four days prior to the start of a growth inhibition experiment, a new algal culture was prepared and allowed to grow on a shaking table at 20 +/- 1°C in continuous light (70 µE/(m².s)).

ACCLIMATION
no data

Test type:

not specified

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

No data

Test temperature:

25°C

pH:

7.4

Dissolved oxygen:

No data

Salinity:

Not applicable

Nominal and measured concentrations:

Nominal concentrations: 3.2, 5.6, 10, 18 and 32 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: no data
- Initial cells density: 10E4 algal cells/mL
- Control end cells density: no data
- No. of vessels per concentration (replicates): 3 replicates + 1 background concentration (no algae added)
- No. of vessels per control (replicates): 3 replicates + 1 background concentration (no algae added)
No further data.

GROWTH MEDIUM
- Standard medium used and composition: See "Details on test organisms / method of cultivation"

TEST MEDIUM / WATER PARAMETERS
- Source: OECD medium
No further data

OTHER TEST CONDITIONS
- Sterile test conditions: no data
- Adjustment of pH: The pH of the stock suspensions was adjusted to 7.4 before introduction into the test flasks. The test flasks were not subjected to pH adjustment.
- Photoperiod: no, constant illumination
- Light intensity: 70 µE/(m².s)

EFFECT PARAMETERS MEASURED:
- Determination of cell density: yes, using a cell counter (Beckman Coulter Counter, Gent, Belgium). The average specific growth rate was then calculated as the slope of a linear regression of the natural logarithm of the measured cell density (corrected for background / versus time).
- Chlorophyll measurement: no

TEST CONCENTRATIONS
no data

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: cerium dioxide diameter: 14 nm

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

5.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: cerium dioxide diameter: 14 nm

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

10.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: cerium dioxide diameter: 14 nm

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: cerium dioxide diameter: 20 nm

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

5.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: cerium dioxide diameter: 20 nm

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

11.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: cerium dioxide diameter: 20 nm

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: cerium dioxide diameter: 29 nm

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

5.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: cerium dioxide diameter: 29 nm

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

19.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: cerium dioxide diameter: 29 nm

Details on results:

- Characterisation of nanoparticles:
The suspensions appear turbid within an hour of suspension preparation, indicating particle aggregation to diameters ranging from 462 to 552 nm. Hence, the organisms were exposed to cerium oxide nanoparticles aggregates.

- Biological results:
Toxicity was observed from 5.6 mg/L cerium dioxide nanoparticles for all nanoparticles sizes (14, 20 and 29 nm).

Results of the five hypotheses to explain the observed algal toxicity:

1) The toxicity was an artefact due to clustering of cerium dioxide nanoparticles with algal cells: identical results were obtained using both algal cell density measurement methods (i.e. cell number measurements and fluorescence spectroscopy of extracted chlorophyll) at low cerium dioxide concentrations. However, at the highest test concentrations of 18 and 32 mg CeO₂ / L a large discrepancy exists; i.e., only little or no chlorophyll was detected. In conclusion, decreased algal cell density was not due to clustering. In case of severe clustering of algal cells and cerium dioxide in an algal growth inhibition experiment, one would expect lower toxicity when the algal cell density is analysed using chlorophyll extraction. However, based on the experiments performed, the authors have no explanation for the absence of chlorophyll at higher test concentrations.

2) Cerium dioxide nanoparticles were taken up or adsorb to the algal cell wall: the TEM study revealed only weak physical interaction between single nanoparticles or aggregates and algal cells. No convincing images of either uptake or strong adsorption to the cell wall were found.

Remark: However, the results of these hypotheses are contradictory with the observation reported in the Daphnia reproduction test presented in the same publication. Indeed, in this last experiment, it is indicated that when cerium dioxide nanoparticles agglomerates were mixed with the algal cells, both clustered together and sedimented.

3) The toxicity was due to dissolved cerium: the concentration of dissolved cerium in all samples in the OECD algae medium was beneath the detection level of the ICP-MS analysis. This indicates that dissolution of the cerium dioxide nanoparticles in the test medium is negligible. It can be ruled out that dissolved cerium was the cause of toxicity in these experiments.

4) The toxicity was due to nutrition depletion: the first experiment showed no adsorption of ammonium, but adsorption of phosphate to the particle surface was observed to a large extent; around 50% in the 32 mg/L 14 nm cerium dioxide suspensions. The adsorption measured after 24 h and 72 h was identical and depended on cerium dioxide concentration and nominal particle diameter. In the second experiment it was shown that a 50% reduction of phosphate in the OECD algae medium had no significant effect on the algal growth rate. Reduction in algal growth due to phosphate deficiency was observed only when 60% of the PO43- was depleted. A 50% reduction in growth rate was observed in phosphate free medium. As a consequence, this hypothesis was not valid. However, it can not be ruled out that physicochemical changes occurred in the liquid interface between cerium dioxide nanoparticles and algal cells.

Remark: A statistically significant phosphate depletion due to complexation with cerium dioxide was already observed in the RCC study (2007) on the bulk form. Therefore, such process seems usual with cerium dioxide. The potential indirect effect of phosphate depletion on algal growth rate cannot be definitely ruled out.

5) The toxicity was due to shading: no evidence of shading was found. When 14 nm cerium dioxide nanoparticles and algal cells were spatially separated in such a way that light had to cross a suspension of cerium dioxide nanoparticles before reaching the algal cells, no decrease in algal cell density was observed. Consequently, this hypothesis was unvalid.

In summary, none of these hypotheses can unambiguously be considered as valid following experimental testing. However, it should be kept in mind that clustering between algal cells and nanoparticles and indirect effect of phosphate depletion cannot be completely ruled out due to weight of evidence brought by other studies (see remarks in italic).

Validity criteria fulfilled:

not specified

Conclusions:

Cerium dioxide nanoparticles appeared harmful to Pseudokirchneriella subcapitata under the tested conditions.

Executive summary:

The 72hr-acute toxicity of cerium dioxide nanoparticles to Pseudokirchneriella subcapitata was studied according to the OECD Guideline for Testing of Chemicals, n°201 (2006). Algae were exposed to test chemicals at nominal concentrations of 3.2, 5.6, 10, 18 and 32 mg/L for 72 hr at three nanoparticles sizes (14, 20 and 29 nm). The cell density was measured daily and the average specific growth rate was calculated.

Toxicity was observed from 5.6 mg/L cerium dioxide nanoparticles for all nanoparticle sizes (14, 20 and 29 nm). And the following 72h-NOEC and -EC₅₀ can be deduced on the basis of growth rate inhibition:

14-nm cerium dioxide nanoparticles:

72h-NOEC (Pseudokirchneriella subcapitata) = 3.2 mg/L

72h-EC₅₀ (Pseudokirchneriella subcapitata) = 10.2 mg/L

20-nm cerium dioxide nanoparticles:

72h-NOEC (Pseudokirchneriella subcapitata)= 3.2 mg/L

72h-EC₅₀(Pseudokirchneriella subcapitata) = 11.7 mg/L

29-nm cerium dioxide nanoparticles:

72h-NOEC (Pseudokirchneriella subcapitata)= 3.2 mg/L

72h-EC₅₀(Pseudokirchneriella subcapitata) = 19.1 mg/L

To explain the observed toxicity, the authors tested several hypotheses: toxicity was due to: (1) an artefact of measurement, (2) adsortpion to the algal cells walls, (3) dissolved cerium, (4) nutriment depletion, (5) shading. However, none of these hypotheses can unambiguously be considered as valid. Nevertheless, it should be kept in mind that clustering between algal cells and nanoparticles and indirect effect of phosphate depletion cannot be completely ruled out due to weight of evidence brought by other studies.

Thus, cerium dioxide nanoparticles appear harmful to Pseudokirchneriella subcapitata under the tested conditions, regardless the particle size.

Reference 4

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From 11-SEP-2006 to 12-SEP-2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Triplicate samples from each test medium (loading rate = 100 mg/L and dilutions 1:2, 1:4, 1:8, 1:16, 1:32) and from the control were taken just before the start of the test and after 24, 48 and 72 hours. However, the cerium concentrations were determined only in the control, in undiluted filtrate and dilution 1:2, as the other dilutions were below the 72h-NOELR, and thus were not relevant for interpretation.
- Sampling method: data not available
- Sample storage conditions before analysis: Immediately after sampling, the samples were filtered through glass fibre microfilters and acidified with 3% (v/v) nitric acid to stabilise the test item during the storage period. Then the samples were deep-frozen and stored at about -20°C until analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: An undiluted filtrate of a supersaturated dispersion with the loading rate of 100 mg/L and the dilutions 1:2, 1:4, 1:8, 1:16, 1:32 of the filtrate were tested. The dispersion with the loading rate of 100 mg/L was prepared by dispersing 181.1 mg of the test item in 1800 mL of test water. The test item was mixed into the test water as homogeneously as possible using ultrasonic treatment for 15 min and intense stirring. The dispersion was stirred on a magnetic stirrer at room temperature in the dark for seven days. After the stirring period, the dispersion was filtered through a membrane filter (0.45 µm). The undiluted filtrate with the maximum concentration of dissolved test item was used as the highest concentrated test medium. The test medium was prepared just before the start of the test.
- Eluate: no
- Differential loading: yes
- Controls: blank (test water without test item)
- Evidence of undissolved material (e.g. precipitate, surface film, etc): yes, on the bottom of the stirring vessel, but not in the final test solution

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Scenedesmus subspicatus CHODAT
- Strain: No. 86.81 SAG
- Source: supplied by the Collection of Algal Cultures (SAG, Institut for Plant Physiology, University of Göttingen, D-37073 Göttingen, Germany)
- Age of inoculum (at test initiation): The algae were taken from an exponentially growing pre-culture, which was set up three days prior to the test under the same conditions as in the test.
- Method of cultivation: Under standardised conditions according to the test guidelines: The algae were cultivated and tested in synthetic test water (analytical grade salts dissolved in sterile purified water).


ACCLIMATION
- Acclimation period: three days
- Culturing media and conditions: same as in the test
- Any deformed or abnormal cells observed: data not available

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

none

Hardness:

0.24 mmol/L (= 24 mg/L as CaCO3)

Test temperature:

23 to 24°C

pH:

At the start of the test, the pH values in the test media and the control ranged from 7.9 to 8.3.
At the end of the test, pH values of 8.1 to 8.5 were measured.

Dissolved oxygen:

not measured

Salinity:

not applicable

Nominal and measured concentrations:

nominal concentrations: undiluted filtrate (loading rate: 100 mg/L), dilution 1:2 (loading rate 50 mg/L), dilution 1:4 (loading rate 25 mg/L), dilution 1:8 (loading rate 12,5 mg/L), dilution 1:16 (loading rate 6.25 mg/L), dilution 1:32 (loading rate 3.125 mg/L)

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type: Erlenmeyer flasks covered with glass dishes
- Material, size, headspace, fill volume: 50 mL, filled with 15 mL of algal suspension
- Aeration: no, but algal suspensions were continuously stirred by magnetic stirrers.
- Type of flow-through (e.g. peristaltic or proportional diluter): none (static test)
- Renewal rate of test solution (frequency/flow rate): not applicable (static test)
- Initial cells density: 5000 algal cells per mL of test medium
- Control end cells density: 593000 cells/mL
- No. of vessels per concentration (replicates): three replicates
- No. of vessels per control (replicates): six replicates

GROWTH MEDIUM
- Standard medium used: yes, the algae were cultivated in synthetic test water, prepared according to the test guidelines. Analytical grade salts were dissolved in sterile purified water.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: sterile purified water
- Total organic carbon, Particulate matter, Metals, Pesticides, Chlorine, Alkalinity, Ca/mg ratio, Conductivity: data not available
- Culture medium different from test medium: no
- Intervals of water quality measurement: The pH was measured and recorded in the test concentration and the control at the start and at the end of the test. The water temperature was measured and recorded daily in an Erlenmeyer flask filled with water and incubated under the same conditions as the test flasks. The appearance of the test medium was also recorded daily. The concentration of phosphate was determined in the test medium and the control at the start of the test and then daily until the end of the test using a photometric method (Merck Spectroquant phosphate test 1.14842.0001). Prior to the determination, the algal cells were removed by filtration trough glass fibre microfilters (GF/C Whatman with a maximum pore size of about 1.2 µm).

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuously illuminated
- Light intensity and quality: The measured light intensity was about 7000 Lux (mean value) and was achieved by fluorescent tubes installed about 35 cm above the test flasks.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Determination of cell concentrations: Small volumes of the test media and the control (1.0 mL) were taken out of all test flasks after 24, 48 and 72 hours of exposure, and were not replaced. The algal cell densities in the samples were determined by counting with an electronic particle counter (Coulter Counter, Model ZM), with at least two measurements per sample. The measurements were performed at least in duplicate. Inhibition of algal growth was determined from: (i) the area under the growth curves (AUC) (= biomass integral), (ii) the specific growth rates (µ), and (iii) the yield (Y).
- Other: In addition, after 72 hours exposure, a sample was taken from the control and from a test concentration (dilution 1:2). The shape and size of the algal cells were examined microscopically in these samples.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Justification for using less concentrations than requested by guideline: not applicable
- Range finding study: yes
- Test concentrations: not reported
- Results used to determine the conditions for the definitive study: not reported

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: not determined

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

91 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 86-94

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

BIOLOGICAL RESULTS & INFLUENCE OF PHOSPHATE CONCENTRATIONS
- Exponential growth in the control: yes (in the control, the cell density increased by a factor of 119 over 72 hours)
- Observation of abnormalities: no, the shape and size of the algal cells was not affected

A statistically significant inhibitory effect was reported on the growth of Scenedesmus subspicatus after the test period of 72 h at the loading rate of 100 mg/L. Thus, this loading rate was determined as the 72h-LOELR. The 72h-NOELR was determined to be the loading rate of 50 mg/L. The 72h-EL50 was set superior to 100 mg/L.The inhibition of algal growth in the highest test concentration was presumably caused by a secondary effect, the complexation of the essential algal nutrient phosphate by the test item. The measured concentration of phosphate in the undiluted filtrate of the dispersion stirred for seven days was much lower than in the test water. A statistically significant decrease of the phosphate concentration was determined at the loading rate of 100 mg/L. Thus, the growth inhibition determined at this loading rate, which corresponded to the maximum concentration of dissolved test item, may have been caused by depletion of phosphate in the test medium, rather than by a toxic effect of cerium dioxide. As a consequence, it seems that cerium dioxide should not have a harmful effect on the algae up to its solubility limit into water.

APPEARANCE OF THE TEST MEDIUM
No remarkable observations were made concerning the appearance of test media. The test media of all test concentrations were clear solutions throughout the entire test period.

ANALYTICAL MONITORING
The analytically measured concentrations of cerium in the test media samples (dilutions 1:2 and in the undiluted filtrate) taken at the start, at days 1 and 2 and the end of the test were below the limit of quantification (LOQ = 1 µg/L).

Results with reference substance (positive control):

- Results with reference substance valid? yes
- 72-hr EC50 for the growth rate = 1.0 mg/L (acceptance range: 0.44-1.16 mg/L) (potassium dichromate)

Reported statistics and error estimates:

The EL10 and EL50 values for the different growth parameters and their 95% confidence intervals were calculated as far as possible by Probit Analysis.
For the determination of the LOELR and NOELR, the calculated mean AUC, the mean growth and the mean yield at the test concentrations were compared to the corresponding control values by multiple Dunnett-tests.

Table 1: Algal cell density during the 72-hour test period

Dilution (loading rate)	Loading rate # (mg/L)		Density of algal cells (cell number x 10,000/mL) after
			24 h	48 h	72 h
Control	-	m s n	2.7 0.2 6	10.1 1.0 6	59.3 2.7 6
Dilution 1:32	3.1	m s n	2.6 0.2 3	10.8 1.3 3	59.0 1.7 3
Dilution 1:16	6.3	m s n	2.5 0.2 3	11.6 0.9 3	59.7 1.6 3
Dilution 1:8	12.5	m s n	2.6 0.1 3	10.1 0.9 3	58.9 6.4 3
Dilution 1:4	25	m s n	2.6 0.1 3	11.4 0.3 3	63.4 1.1 3
Dilution 1:2	50	m s n	2.6 0.0 3	11.6 0.2 3	62.9 4.7 3
Undiluted filtrate	100	m s n	2.7 0.1 3	11.1 0.7 3	28.4 1.5 3

m: mean value; s: standard deviation; n: number of flasks

 

Table 2: Areas under the Growth Curves (AUC) and percentage inhibition of AUC (I [AUC]) during the test period

Dilution	Loading rate # (mg/L)	Areas under the growth curves AUC and % inhibition of AUC
		0 to 24 h		0 to 48 h		0 to 72 h
		AUC	I [AUC] (%)	AUC	I [AUC] (%)	AUC	I [AUC] (%)
Control	-	1.10	0.0	7.00	0.0	41.18	0.0
1:32	3.1	1.04	5.3	7.23	-3.3	41.61	-1.0
1:16	6.3	0.98	11.4	7.48	-6.7	42.61	-3.5
1:8	12	1.04	5.3	6.88	1.7	40.88	0.7
1:4	25	1.03	6.1	7.51	-7.2	44.40	-7.8
1:2	50	1.05	4.5	7.63	-9.0	44.35	-7.7
Undiluted filtrate	100	1.10	0.0	7.50	-7.1	26.76*	35.0

AUC x 5000

-% inhibition: increase in growth relative to that of control

*: mean value significantly lower than in the control (according to Dunnett’s tests, one-sided, α = 0.05)

 

Table 3: Growth rates r and percentage inhibition of r (Ir) during the test period

Dilution (loading rate)	Loading rate # (mg/L)	Growth rate r and % of inhibition of r
		0 to 24 h		0 to 48 h		0 to 72 h
		r (1/day)	lr (%)	r (1/day)	lr (%)	r (1/day)	lr (%)
Control	-	1.68	0.0	1.50	0.0	1.59	0.0
1:32	3.1	1.64	2.5	1.53	-2.2	1.59	0.1
1:16	6.3	1.59	5.7	1.57	-4.5	1.59	-0.2
1:8	12	1.64	2.5	1.50	0.0	1.59	0.2
1:4	25	1.64	2.9	1.56	-4.1	1.61	-1.4
1:2	50	1.65	2.1	1.57	-4.6	1.61	-1.2
Undiluted filtrate	100	1.69	-0.1	1.55	-3.2	1.35*	15.4

-% inhibition: increase in growth relative to that of control

*: mean value significantly lower than in control (according to Dunnett-test, one sided smaller, α = 0.05)

 

Table 4: Yield (y) and percentage inhibition of y (Iy) during the test period

Dilution (loading rate)	Loading rate # (mg/L)	Yield y and inhibition of y
		0 to 24 h		0 to 48 h		0 to 72 h
		yield	ly (%)	yield	ly (%)	yield	ly (%)
Control	-	2.20	0.0	9.61	0.0	58.75	0.0
1:32	3.1	2.08	5.3	10.30	-7.2	58.45	0.5
1:16	6.3	1.95	11.4	11.05	-15.0	59.22	-0.8
1:8	12	2.08	5.3	9.60	0.1	58.38	0.6
1:4	25	2.07	6.1	10.88	-13.3	62.90	-7.1
1:2	50	2.10	4.5	11.07	-15.2	62.37	-6.2
Undiluted filtrate	100	2.20	0.0	10.60	-10.3	27.92*	52.5

y x 5000

-% inhibition: increase in growth relative to that of control

*: mean value significantly lower than in control (according to Dunnett-test, one-sided, α = 0.05)

Validity criteria fulfilled:

yes

Remarks:

In the control, the cell density increased by a factor of 119 during the test period of 72 hours (> 16), the coeff. of variation of the daily growth rates was 15.7% (< 35%), and the coeff. of variation of the average specific growth rates was 1.0% (< 7%).

Conclusions:

A statistically significant inhibitory effect was reported on the growth of Scenedesmus subspicatus after the test period of 72 h at the loading rate of 100 mg/L. Thus, this loading rate was determined as the 72h-LOELR. The 72h-NOELR was determined to be the loading rate of 50 mg/L. The 72h-EL50 was set superior to 100 mg/L. However, a significant phosphate depletion was observed at the highest tested concentration, probably due to a complexation process with the test item. As a consequence, the reduction of growth here observed was probably caused by an indirect effect of phosphate lack, rather than a toxic effect of cerium dioxide.

Executive summary:

The influence of cerium dioxide on the growth of the green algal species Scenedesmus subspicatus was investigated in a 72-hour static test according to guidelines EU C.3 (1992) and OCDE 201 (2006). The GLP were stated.

Due to the low water solubility of the test item, a supersaturated dispersion with the loading rate of 100 mg/L was prepared. Then the dispersion was filtered and the undiluted filtrate and the dilutions 1:2, 1:4, 1:8, 1:16 and 1:32 were used as test media. Additionally, a control was tested in parallel.

A statistically significant inhibitory effect was reported on the growth of Scenedesmus subspicatus after the test period of 72 h at the loading rate of 100 mg/L. Thus, this loading rate was determined as the 72h-LOELR. The 72h-NOELR was determined to be the loading rate of 50 mg/L. The 72 h-EL50 was set superior to 100 mg/L. The inhibition of algal growth in the highest test concentration was presumably caused by a secondary effect, the complexation of the essential algal nutrient phosphate by the test item. The measured concentration of phosphate in the undiluted filtrate of the dispersion stirred for seven days was much lower than in the test water. A statistically significant decrease of the phosphate concentration was determined at the loading rate of 100 mg/L. Thus, the growth inhibition determined at this loading rate, which corresponded to the maximum concentration of dissolved test item, may have been caused by depletion of phosphate in the test medium, rather than by a toxic effect of cerium dioxide. As a consequence, it seems that cerium dioxide should not have a harmful effect on the algae up to its solubility limit into water.

Reference 5

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Remarks:

Some tables and figures of the publication report results on bulk cerium dioxide. However, the article primarily deals with the nanoparticle form, and no experimental detail is given concerning the experiment with the micrometric form. Therefore, a reliability 4 was attributed to this data.

Reason / purpose for cross-reference:

reference to same study

Principles of method if other than guideline:

No data are available on the bulk form. As results are presented concomittantly with those on nanoparticles, one can expected that the same experimental protocol was applied (i.e. OECD method 201).

GLP compliance:

not specified

Analytical monitoring:

not specified

Details on sampling:

No data on the analytical monitoring of concentrations of the bulk cerium dioxide during the test.

Vehicle:

not specified

Details on test solutions:

No data are available on the bulk form.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

No data are available concerning the organisms tested during the experiment on the bulk form.

Test type:

not specified

Water media type:

freshwater

Total exposure duration:

72 h

Hardness:

No data

Test temperature:

No data

pH:

No data

Dissolved oxygen:

No data

Salinity:

Not applicable

Nominal and measured concentrations:

No data are available on the bulk form.

Details on test conditions:

No data are available on the bulk form.

Reference substance (positive control):

not specified

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Validity criteria fulfilled:

not specified

Conclusions:

Based on the limited details provide, cerium dioxide (micrometric form) does not present algal toxicity.

Executive summary:

In this publication primarily dealing with nanoparticulate cerium dioxide, some results are given concerning the bulk form. A 48-hour NOEC superior to 1000 mg/L based on growth was reported, implying that bulk cerium dioxide does not present toxicity for the algae Pseudokirchneriella subcapitata.

Reference 6

Endpoint:

toxicity to aquatic algae and cyanobacteria

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

Remarks:

No analytics were performed. No international guideline was used for the present study. Little information regarding the material and methods used.

Qualifier:

no guideline followed

Principles of method if other than guideline:

The growth parameter of green algae (Chlorella vulgaris) is studied by inoculating cells on agar plate containing mineral salts supplemented with the test material. After 12-15 days, the percentage survival of algae was assessed by colony count. To check whether the death of the green algae was caused by the testing material toxicity or by lack of phosphate, another experiment was performed by treating the cells with phosphate-supplemented and phosphate free basal media.

GLP compliance:

not specified

Analytical monitoring:

not specified

Details on sampling:

Only sampling to assess phosphate removal by ZrOCl2. After complete precipitation, the medium was centrifuged and the supernatant analysed.

Vehicle:

no

Details on test solutions:

No information available

Test organisms (species):

Chlorella vulgaris

Details on test organisms:

TEST ORGANISM
- Common name: Chlorella vulgaris Beijerinck

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

15 d

Hardness:

no data

Test temperature:

no data

pH:

no data

Dissolved oxygen:

no data

Salinity:

not applicable

Nominal and measured concentrations:

Nominal concentrations: 20, 40, 60, 80, 100 and 200 mg/L

Details on test conditions:

The effect of ZrOCl2 on the growth of Chlorella vulgaris Beijerinck was studied by inoculating 14 x 10+4 cells on agar containing mineral salts supplemented with 20, 40, 60, 80, 100 or 200 mg/L grade of ZrOCl2. After 12-15 days, the percentage survival of algae was assessed by colony count.
TEST SYSTEM
- Test vessel: agar plates
- Initial cells density: 14 x 10+4 cells

GROWTH MEDIUM
Agar plates containing mineral salts

OTHER TEST CONDITIONS
- Adjustment of pH: yes

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Determination of cell concentrations: growth was determined by optical density

Reference substance (positive control):

no

Duration:

15 d

Dose descriptor:

NOEC

Effect conc.:

> 200 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

by optical density

Remarks on result:

other: medium supplemented with phosphates

Details on results:

ZrOCl2 precipitated phosphate and consequently adversely affected algal growth from the lowest concentration used (20 ppm) up to 100 ppm at a pH range of 2-11. An experiment with phosphate-supplemented test medium (phosphate in excess of Zr) however did not display any adverse effect on growth at (nominal) ZrOCl2 concentrations of 100 and 200 mg/L. Therefore, ZrOCl2 does not seem to be harmful to algae and the observed effects can be ascribed to phosphate deprivation.

Validity criteria fulfilled:

not applicable

Conclusions:

Growth inhibition of Chlorella vulgaris was attributed to the unavailibility of phosphate. Therefore, zirconium dichloride oxide was not toxic at up to 200 mg/L only if phosphate is added in the culture medium of algae.

Executive summary:

By treating Chlorella cells with ZrOCl2, growth rate (optical density measurement) was inhibited and started at the lowest concentration used (20 mg/L). However, the reduction of growth was due to the lack of phosphate precipitated by the ZrOCl2. In fact, an experiment performed by treating the cells with 100 mg/L and 200 mg/L of ZrOCl2 in phosphate-supplemented medium, displayed no impact on growth rate on Chlorella sp. Therefore, the growth inhibition which was observed, was considered due to the unavailability of phosphate and not to zirconium toxicity. The NOEC value is assessed at > 200 ppm of ZrOCl2.

Reference 7

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

5 August - 1 October 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: control and each test group at 0 and 72 h for zirconium analysis, and at 0, 24, 48 and 72 h for phosphate analysis.
- Sampling method: replicates were pooled
- Sample storage conditions before analysis: -20°C

Vehicle:

no

Details on test solutions:

A saturated solution was prepared by dispersion of 1100 mg test item in 11 liters of culture medium with the aid of a propeller stirrer at 1500 rpm at ca. 21°C for 24h. The undissolved test item was removed by filtration through a 0.2 µm Gelman Acrocap filter to give 100% (v/v) saturated solution.
A series of dilutions was made from this 100% v/v stock solution to give further stock solutions of 32, 10, 3.2 and 1.0% v/v saturated solution. An aliquot of 900 ml of each stock solution was separately inoculated with 4 ml algal suspension.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Strain: CCAP 276/20
- Source (laboratory, culture collection): Liquid cultures obtained from the Culture Collection of Algae and Protozoa (CCAP), Dunstaffnage Marine Laboratory, Oban, Argyll, Scotland. Master cultured were maintained in the lab by periodic replenishment of culture medium.
- Method of cultivation: Under constant aeration and constant illumination at 21+/-1°C

ACCLIMATION
- Acclimation period: 100 ml volumes of culture media containing an initial cell density of 1000 cells/ml were constantly shaked (100-150 rpm) and illuminated at 24+/-1°C until the density was 10,000-100,000 cells/ml.
- Culturing media and conditions (same as test or not): same as test
- Any deformed or abnormal cells observed: no

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

no data

Test temperature:

24+/-1°C

pH:

At 0 h: 7.8-7.9 (control) and 7.6-7.8 (test concentrations)
At 72h: 7.8 (control) and 7.7-7.8 (test concentrations)

Dissolved oxygen:

no data

Salinity:

not applicable

Nominal and measured concentrations:

Nominal (% v/v saturated solution or mg/L): 0, 1.0, 3.2, 10, 32, 100
Measured: <0.010 mg Zr/L (LOQ) in all test solutions

Details on test conditions:

TEST SYSTEM
- Test vessel: conical flasks
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 250 ml glass conical flasks containing 100 ml of solution
- Aeration: flasks were plugged with polyurethane foam bungs and shaken at 150 rpm
- Initial cells density: 4000 cells/ml
- Control end cells density: 142,000 cells/ml (mean of 6 flasks)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reverse osmosis purified deionised water
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Adjustment of pH: to 8.1+/-0.1 of the culture medium
- Photoperiod: continuous
- Light intensity and quality: 7000 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Coulter multisizer particle counter

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study
- Test concentrations: 0.10, 1.0, 10 and 100% v/v saturated solution
- Results used to determine the conditions for the definitive study: reduced growth at 10 and 100% v/v saturated solution.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no abnormalities observed
- Colour differences: at the start of the test all cultures were clear colourless solutions. After 72h all cultures were very pale green dispersions, while the 100% saturated solution was extremely pale green.
- Any stimulation of growth found in any treatment: no
- Effect concentrations exceeding solubility of substance in test medium: no

Results with reference substance (positive control):

ErC50 (0-72h): 0.74 mg/L and NOErC: 0.25 mg/L. The results were within the normal ranges.

Reported statistics and error estimates:

Inhibition of growth rate: No statistically significant differences between control and test solutions (P>=0.05) except for the 100% v/v saturated solution (P<0.05).

Analysis of phosphate:

At 0 hours, phosphate concentration decreased with increasing test concentration:

	Phosphate concentration (mg/L) at 0h	24h	48h	72h
control	1.19	1.12	0.846	0.0393
1.0%	1.18	1.02	0.924	<LOQ
3.2%	1.13	0.999	0.881	0.116
10%	1.04	0.959	0.735	<LOQ
32%	0.798	0.738	0.481	<LOQ
100%	0.116	0.0718	<LOQ	<LOQ

A similar concentration dependent pattern was observed at 24, 48 and 72 hours, with measured phosphate concentrations for all but the 3.2% saturated solution being less than the LOQ (0.021 mg/L). In the control, phosphate decreased from 1.19 mg/L at 0 h to 0.039 mg/L at 72 h. The decrease in phosphate concentration during the test was due to the use of phosphate for algal growth.

The reduced level of phosphate (compared to control) shown already before the start of the test, which is statistically significant at the highest saturated concentration, was possibly the cause for the reduced algal growth rather than true toxicity of the test compound.

Validity criteria fulfilled:

yes

Conclusions:

The effect of zirconium basic carbonate on the growth of Desmodesmus subspicatus has been investigated over a 72h period. As the substance could not be detected ( 100 mg/L and the NOErC was 32 mg/L. Reduced growth rate was concurrent with phosphate depletion due to complexation with zirconium and precipitation. Therefore, the growth inhibition which was observed, was considered due to the unavailability of phosphate and not to zirconium toxicity.

Reference 8

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

22 November 2012 - 27 November 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

A sample of each test concentration was taken for chemical analysis at 0 and 72 hours in order to determine the stability of the test item under test conditions. All samples were stored frozen prior to analysis.
The test samples were thawed with the aid of sonication. Nitric acid (4 mL) was added to a volume (200 mL) of sample and the samples were sonicated for 15 minutes before being filtered through 0.45 µm cellulose acetate filters.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
Method: Due to the low aqueous solubility of the test item, a dispersion was prepared by adding 100 mg of test item to the surface of 2 liters of culture medium and stirred vigorously using a magnetic stirrer for 10 minutes after which the pH was adjusted from 6.3 to 7.5. The media was then stirred for a further 24 hours. After stirring, any undissolved test item was removed by filtration through a 0.2 µm Gelman Acrocap filter (initial 500 mL discarded) to give a 100% v/v saturated stock solution. The test item used represents a solution containing 29.2% anhydrous zirconium dichloride oxide. Given the low aqueous solubility it was considered that a loading rate of 50 mg test item/L, which corresponds to 14.6 mg zirconium dichloride oxide/L, was sufficient to ensure 100% saturation of the test media. A series of dilutions was made from the 100% v/v saturated solution to give further stock solutions of 10, 1.0 and 0. 10% v/v saturated solution. An aliquot (450 mL) of each of the stock solutions was separately inoculated with algal suspension (3.9 mL).

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata strain
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland.
- Method of cultivation: Prior to the start of the test sufficient master culture was added to approximately 100 mL volumes of culture media contained in conical flasks to give an initial cell density of approximately 1000 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100–150 rpm) and constant illumination at 24 ± 1 °C until the algal cell density was approximately 10,000-100,000 cells/mL.

ACCLIMATION
- Acclimation period: The master cultures were maintained in the laboratory under constant aeration and constant illumination at 21 ± 1 °C.
- Culturing media and conditions (same as test or not): same as test
- Any deformed or abnormal cells observed: no

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

no data

Test temperature:

24± 1°C

pH:

Control:
0 h: 7.7
72 h: 8.0
Treatments:
0 h: 7.5-7.7
72 h: 7.8-8.0

Dissolved oxygen:

not applicable

Salinity:

not applicable

Nominal and measured concentrations:

Nominal (% v/v saturated solution): 0, 1.0, 10, 100
Measured: 100% v/v saturated solution: < 0.011 mg Zr/L (LOQ)

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL conical flasks
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 250 mL glass conical flasks each containing 100 mL of test preparation and plugged with polyurethane foam bungs to reduce evaporation
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: yes: to 7.5
- Light intensity and quality: approximately 7000 lux provided by warm white lighting (380–730 nm)
- Shaking: constantly shaken at approximately 150 rpm for 72 hours

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Determination of cell concentrations: Coulter® Multisizer Particle Counter

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Range finding study: yes
- Test concentrations: 0.10, 1.0, 10 and 100% v/v saturated solution.
- Results used to determine the conditions for the definitive study: Chemical analysis of the 10 and 100% v/v saturated solution test preparations at 0 and 72 hours showed that dissolved zirconium concentrations were less than the limit of quantitation, which was determined to be 0.011 mg Zr/L. As no zirconium was in solution, it was considered unnecessary to continue to the definitive stage of testing.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

80 other: % v/v saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

10 other: % v/v saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no abnormalities observed
- Any stimulation of growth found in any treatment: no
- Observed effects: In the 100% v/v saturated solution, 73% inhibition (growth rate-based) was observed after 72 h. The EC50 and NOEC based on growth rate were reported to be 80% and 10% v/v saturated solution, respectively. However, dissolved zirconium appeared to be < LOQ (i.e. 11 µg Zr/L) in all of the treatments, indicating that the observed inhibition of growth may be due to a secondary effect.

Results with reference substance (positive control):

ErC50 (0 – 72 h): 1.1 mg/L; 95% confidence limits 1.0 – 1.3 mg/L
EbC50 (0 – 72 h): 0.70 mg/L
The results from the positive control with potassium dichromate were within the normal ranges for this reference item.

Reported statistics and error estimates:

For each test concentration % inhibition was plotted against test concentration (the latter on a logarithmic axis) and a line fitted by computerized interpolation using the Xlfit software package (IDBS). ECx values were then determined from the equation for the fitted line.
The NOEC was determined by visual inspection of the growth rate and yield data.

Validity criteria fulfilled:

yes

Conclusions:

In this study, it was impossible to keep zirconium dissolved at measurable levels under the conditions of the test (i.e., dissolved Zr < LOQ = 11 µg Zr/L). However, after 72 h of exposure of Pseudokirchneriella subcapitata to a 100% v/v saturated solution, significant growth inhibition was observed, resulting in a 72-h ErC50 and NOErC of 80 and 10% v/v saturated solution. Because no bioavailable (dissolved) zirconium was detected in the test media, the observed toxicity may be rather due to phosphate deprivation (see other studies) which is a secondary effect and not considered environmentally relevant.

Reference 9

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Justification for type of information:

Read across from studies performed with zirconium dichloride oxide and zirconium basic carbonate. The read across justification document is attached in IUCLID Section 13.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Remarks on result:

other: Zirconium dioxide is not considered to be toxic or harmful to aquatic algae.

Remarks:

This conclusion was based on the results of studies performed with the read across substances zirconium dichloride oxide (which is a 'water soluble' zirconium compound) (Vryenhoef, 2014; Kumar and Rai, 1978) and zirconium basic carbonate (which is a sparingly soluble zirconium compound) (Vryenhoef and Mullee, 2010). Any observed adverse effects were concurrent with phosphate depletion from the test medium (due to complexation with zirconium) and therefore the observed effects are attributed to the secondary effect of phosphate deprivation. This may occur in confined test systems where limited volumes are used but this is not considered to be an environmentally relevant effect. Together with the information on micrometric and nanometric cerium dioxide as well as the study with a representative nanoform of the reaction mass, it is not expected that the reaction mass of cerium dioxide and zirconium dioxide would cause environmentally relevant harmful effects in algae.

Description of key information

A GLP-compliant study, scored as Klimisch 1 and flagged as a key study, is available on the reaction mass, giving a 72h-EL50 >100 mg/L (Peither, 2008). Some effects were observed at 100 mg/L, but these appeared to be attributed to phosphate depletion from the test medium due to complexation with the test material (phosphate complexation (and precipitation) is a known behaviour of both cerium and zirconium) and are therefore not considered environmentally relevant. The test was carried out with a representative nanoform of the test substance, however the same conclusion is expected for any potential bulk forms of the reaction mass of cerium dioxide and zirconium dioxide, should such forms be placed on the market.

Additionally, supporting information was added to the dossier for the constituents zirconium (dioxide) and cerium dioxide. For zirconium, two studies were included for the read across substance zirconium dichloride oxide, which is a 'water soluble' zirconium compound (Vryenhoef, 2014; Kumar and Rai, 1978), and one study was included with zirconium basic carbonate, which is a sparingly soluble zirconium compound (Vryenhoef and Mullee, 2010). Together these studies confirm that the observed effects are attributed to phosphate depletion from the test medium, which is not considered to be substantial at an environmentally relevant scale. For cerium dioxide, the study of Bätscher (2007b) on micrometric cerium dioxide had similar findings. Van Hoecke et al (2009) studied both micrometric and nanometric cerium dioxide, observing effects on algal growth only for nanometric cerium dioxide samples, but was unable to conclude on the exact mechanism behind this. It could not be excluded that clustering of the nanomaterial with the algae (and consequent sedimentation) as well as phosphate deprivation were responsible for / contributed to the observed effects.

In conclusion, there is no indication that the reaction mass of cerium dioxide and zirconium dioxide, whether nanometric or micrometric, would result in environmentally relevant harmful effects in algae.

Additional information

For toxicity to aquatic algae and cyanobacteria, a study on the reaction mass of cerium dioxide and zirconium dioxide is available (Peither, 2009). Additionally, three studies on zirconium compounds and two studies on cerium dioxide were included as supporting information.

 

In the study of Peither (2009; Klimisch 1), cultures of the green algal species Scenedesmus subspicatus were exposed to a reaction mass of cerium dioxide and zirconium dioxide (containing approximately 60% CeO2 and 30% ZrO2). The NOEC and EC50 values based on growth rate were 32 mg/L and > 100 mg/L respectively (based on nominal concentrations of the reaction mass). Almost no test substance (monitored based on dissolved cerium measurements) was present in the test solutions. The concentration of phosphate was statistically significantly reduced compared to the control in the test solutions. The loss of phosphate can be explained by the formation of insoluble complexes of phosphate with cerium and zirconium (which is a well-known behavior of rare earth elements as well as zirconium in the environment). The observed algal growth inhibition was concurrent with the depletion of phosphate in the test medium and therefore the observed effect was considered a secondary effect and not environmentally relevant.

 

Additionally, read across studies were included for the individual constituents zirconium (dioxide) and cerium dioxide as supporting information.

 

Zirconium substances

A first study was performed using the read across substance zirconium dichloride oxide (Vryenhoef, 2014; Klimisch 1), a 'water soluble' zirconium compound. A range finding experiment indicated that no measurable zirconium could stay in solution (dissolved Zr < LOQ, i.e. < 11 µg Zr/L). In the 100% v/v saturated solution, significant growth inhibition was however observed. The 72-h EC50 and NOEC based on growth rate were 80 and 10% v/v saturated solution, respectively. Because of the absence of bioavailable (dissolved) zirconium in the test media, no further testing was deemed necessary and the observed effect was considered to be a secondary effect (i.e., growth inhibition due to phosphate deprivation).

 

The second study performed with the read across substance zirconium dichloride oxide is the study by Kumar and Rai (1978; Klimisch 2), in which it is shown that algae exposed to zirconium dichloride oxide up to 100 ppm show growth inhibition, especially at 60, 80 and 100 ppm. This effect is caused by precipitation of phosphates which are essential to algae. When algae are supplemented with phosphate in the medium after filtration, growth was comparable to controls. The results suggest that zirconium dichloride oxide is not toxic directly to algae at concentrations up to 100 ppm (nominal concentration, no analytics performed).

Finally, the study of Vryenhoef and Mullee (2010; Klimisch 1) investigated the effect of zirconium basic carbonate (a sparingly soluble zirconium compound) on the growth of Desmodesmus subspicatus over a 72 h period. As zirconium could not be detected (< LOQ) in the test solution, the results were based on nominal concentrations. The ErC50 was > 100 mg/L and the NOErC was 32 mg/L (based on zirconium basic carbonate). Phosphate monitoring during the test indicated that reduced growth rate was concurrent with phosphate depletion due to phosphate complexing with zirconium and precipitation of the formed complexes. The observed effect is clearly a secondary effect which is not considered environmentally relevant.

 

Based on the above, it can be safely concluded that zirconium dioxide (for which no algal growth inhibition studies are available) is not expected to cause any direct zirconium-related toxicity to aquatic plants. This information is supportive of the findings in the study of Peither (2009), which concluded that the observed effects of the reaction mass are most likely due to phosphate deprivation.

Micrometric cerium dioxide

Two experimental studies are available. In the study of Bätscher (2007b; Klimisch 1) a statistically significant inhibitory effect was reported on the growth of Scenedesmus subspicatus after the test period of 72 h at the loading rate of 100 mg/L. The inhibition of algal growth in the highest test concentration was presumably caused by a secondary effect, the complexation of the essential algal nutrient phosphate by the test item. The measured concentration of phosphate in the undiluted filtrate of the dispersion stirred for seven days was much lower than in the test water. A statistically significant decrease of the phosphate concentration was determined at the loading rate of 100 mg/L. Thus, the growth inhibition determined at this loading rate, which corresponded to the maximum concentration of dissolved test item, may have been caused by depletion of phosphate from the test medium, rather than by a toxic effect of cerium dioxide. As a consequence, it seems that cerium dioxide should not have toxic effects on the algae up to its solubility limit into water.

 

The study of Van Hoecke et al. (2009) was given a Klimisch score of 4. In this publication primarily dealing with nanoparticulate cerium dioxide, some results are given concerning the bulk form. A 72-hour NOEC superior to 1000 mg/L based on growth was reported; implying that bulk cerium dioxide does not present toxicity for the algae Pseudokirchneriella subcapitata.

Nanometric cerium dioxide

One publication, scored as Klimisch 2, is available (Van Hoecke et al., 2009). It revealed significant adverse effects of nanoparticulate cerium dioxide on algal growth, with 72h-EC50 and 72h-NOEC in the ranges 10 - 100 mg/L and 1 - 10 mg/L, respectively. To explain the observed toxicity, the authors tested several hypotheses: toxicity was due to: (1) an artefact of measurement, (2) adsorption to the algal cells walls, (3) dissolved cerium, (4) nutriment depletion, (5) shading. However, none of these hypotheses can unambiguously be considered as valid. Nevertheless, it should be kept in mind that clustering between algal cells and nanoparticles and indirect effects of phosphate depletion cannot be completely ruled out due to weight of evidence brought by other studies (see above + for clustering see the study of Van Hoecke et al. (2009) in aquatic invertebrates). Thus, although cerium dioxide nanoparticles appear harmful to Pseudokirchneriella subcapitata under the tested conditions, the observed effects may not be environmentally relevant.