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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
30 November 2007 - 27 February 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
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Triplicate samples were taken from each test medium and from the control just before the start of the test and after 24, 48 and 72 hours. The concentrations of praseodymium were analytically determined in two samples of the dilutions 1:4.6, 1:2.1 and of the saturated solution with the loading rate of 100 mg/L and in two control samples from each sampling date. The samples from the lower test concentrations (dilutions 1:10 and 1:21) were not analysed, since these test concentrations were below the 72-hour NOELR and, thus, not relevant for the interpretation of the biological results.
- Sampling method: data not available
- Sample storage conditions before analysis: Immediately after sampling, the samples were acidified with 10% (v/v) nitric acid to stabilise the test material 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:
According to the results of a pre-experiment (non-GLP), the test material was not completely soluble at the concentration of 100 mg/L in the test water and no homogeneous dispersion could be prepared.
Therefore, a saturated solution of the test material with the loading rate of 100 mg/L and the dilutions 1:2.1, 1:4.6, 1:10 and 1:21 of the saturated solution were tested. Additionally, a control was tested in parallel (test water without test material).
The test method is based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures.
The dispersion with the loading rate of 100 mg/L was prepared by dispersing 200.1 mg of the test material in 2000 mL of test water. The test material was mixed into the test water as homogeneously as possible using ultrasonic treatment for 15 minutes and intense stirring. No auxiliary solvent or emulsifier was used. The dispersion was stirred on a magnetic stirrer at room temperature in the dark for 24 hours. After this stirring period, the stirrer was switched off to allow the undissolved test material to deposit at the bottom of the stirring vessel for 24 hours.
The equilibrated test medium (saturated solution) was carefully separated from the undissolved test material and used as the highest concentrated test medium. Additionally, adequate volumes of the saturated solution were diluted with test water for the preparation of the test media with lower test material concentrations. The test media were prepared just before the start of the test (= addition of algae). The test concentrations were based on the results of a range-finding test and on the results of pre-experiments to determine the water solubility of the test material (non-GLP). The stirring time of 24 hours was chosen according to the results of a pre-experiment in which the maximum concentration of dissolved praseodymium was determined after the stirring time of 24 hours.
- Eluate: no
- Differential loading: yes
- Controls: blank (test water without test material)
- 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
- Age of inoculum (at test initiation): The algal cells 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: Cultivated at the laboratory in synthetic test water, prepared according to the test guidelines. Analytical grade salts were dissolved in sterile purified water.
- Source: supplied by the Collection of Algal Cultures (SAG, Institut for Plant Physiology, University of Göttingen, 37073 Göttingen, Germany)

ACCLIMATION
- Acclimation period: three 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
Post exposure observation period:
no
Hardness:
0.24 mmol/L (= 24 mg/L as CaCO3)
Test temperature:
21 - 22 °C
pH:
At the start of the test, the pH of the test media and the control ranged from 8.2 to 8.6. At the end of the test, pH values between 8.0 and 9.4 were measured. The increase of the pH during the test was caused by the uptake of CO2 by the algae due to their growth.
Dissolved oxygen:
not measured
Salinity:
not applicable
Nominal and measured concentrations:
Nominal loading rates: dilution 1:21 (loading rate 4.8 mg/L), dilution 1:10 (loading rate 10 mg/L), dilution 1:4.6 (loading rate 22 mg/L), dilution 1:2.1 (loading rate 48 mg/L), saturated solution (loading rate: 100 mg/L)
Mean measured concentrations: dilution 1:4.6 (6 μg/L), dilution 1:2.1 (loading rate 17 μg/L), saturated solution (loading rate: 37 μg/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: 371341.5 algal cells per mL of test medium
- 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 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, 48 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: continuously illuminated
- Light intensity and quality: The measured light intensity was about 7600 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 withdrawn daily 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 highest test concentration. The shape and size of the algal cells were examined microscopically in these samples.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.2
- 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: no data
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:
NOELR
Effect conc.:
22 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOELR
Effect conc.:
48 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
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 74 over 72 hours)
- Observation of abnormalities (for algal test): Unusual cell shape, Colour differences, Flocculation, Adherence to test vessels, Aggregation of algal cells: 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 media. All test media were clear solutions throughout the test period.

PHOSPHATE CONCENTRATIONS:
The analysis of the impact of the loading rate on the phosphate amount in the test media showed that the saturated solution had a statistically significant impact on the phosphate concentration in the test medium at all time points (except at 72 hours, where the loss of phosphate over time had a stronger impact on the phosphate concentration than the test material). The dilution 1:2.1 (loading rate of 48 mg/L) had a statistically significant impact on the phosphate concentration at 0 and 48 hours. At the test start, this impact was significant even at the dilution 1:10 (loading rate of 10 mg/L) and above. The loss of phosphate can be explained by the formation of insoluble complexes of phosphate with the test material (which is a well-known behaviour of rare earth elements in the environment) during stirring of the dispersion. The depletion of phosphate in the test medium during the test might have been the reason for the inhibition of algal growth determined at these test concentrations. Thus, growth inhibition due to a secondary effect (i.e. the complexation of the essential algal nutrient phosphate by the test material) cannot be excluded.

ANALYTICAL MONITORING:
The concentrations of praseodymium (Pr) were measured in samples taken daily from the dilutions 1:4.6 (loading rate 22 mg/L), 1:2.1 (loading rate 48 mg/L) and from the saturated solution (loading rate 100 mg/L).
During the test, the concentrations of praseodymium measured in the dilution 1:4.6 ranged from 3 to 7 μg/L, in the dilution 1:2.1 from 13 to 16 μg/L, and in the saturated solution from 24 to 37 μg/L, respectively. The solubility limit reached during this test was thus different from that obtained during the water solubility test (see IUCLID section 4.8). Such contrasting results could be explained by the different water media used in the water solubility test and ecotoxicological studies (media containing analytical grade salts).
Results with reference substance (positive control):
- Results with reference substance valid? yes
- 72 hour 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 EL10 and EL50 values (the respective loading rates of the test material 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 EL90 could not be calculated for AUC and growth rate, as the inhibition of the parameters was below 90% at all test concentrations.
For the determination of the LOELR and NOELR, the 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 /

Dilution

 

Rep. no.

Biomass of algae*

(relative Fluorescence units)

24 hours

48 hours

72 hours

Control

1

2

3

4

5

6

2.7

2.7

2.6

3.0

3.1

3.0

15.6

15.1

17.6

16.5

17.8

16.3

56.7

64.7

61.0

69.6

54.0

59.2

Mean

SD

2.8

0.2

16.5

1.1

60.9

5.6

1:21

1

2

3

2.6

2.8

3.1

17.5

17.8

18.6

71.9

54.4

70.1

Mean

SD

2.8

0.3

18.0

0.5

65.5

9.6

1:10

1

2

3

2.8

3.0

3.0

16.8

17.5

15.3

58.5

56.2

55.8

Mean

SD

3.0

0.1

16.5

1.2

56.8

1.5

1:4.6

1

2

3

2.6

3.2

3.1

14.1

18.6

13.5

55.5

63.6

47.5

Mean

SD

3.0

0.3

15.4

2.8

55.6

8.0

1:2.1

1

2

3

2.4

2.3

2.4

13.2

16.0

14.8

51.0

47.9

44.8

Mean

SD

2.4

0.0

14.7

1.4

47.9

3.1

Saturated solution

1

2

3

2.0

2.4

2.6

7.8

9.3

7.4

8.4

8.3

7.8

Mean

SD

2.3

0.3

8.2

1.0

8.2

0.3

SD: Standard deviation

*: The biomass was determined by fluorescence measurement (duplicate measurements) and is given as relative fluorescence units (x 10³). At the start of the test, the initial cell density was 5000 algal cells/mL, corresponding to 0.82 x 10³ relative fluorescence units).

 

Table 2: Areas under the Growth Curves (AUC)

Treatment / Dilution

Loading rate

(mg/L)

Areas under the growth curves AUC (10³*day)

And inhibition of AUC (IAUC)

0 - 24 hours

0 - 48 hours

0 - 72 hours

AUC

IAUC(%)

AUC

IAUC(%)

AUC

IAUC(%)

Control

-

1.0

0.0

9.9

0.0

47.7

0.0

1:21

4.8

1.0

1.0

10.6

-7.3

51.5

-7.9

1:10

10

1.1

-5.9

10.0

-1.5

45.9

3.9

1:4.6

22

1.1

-5.8

9.4

4.3

44.1

7.6

1:2.1

48

0.8*

22.4

8.5

13.9

38.9*

18.4

Saturated solution

100

0.8*

25.4

5.2*

47.3

12.6*

73.7

*: mean value significantly lower than in the control

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

Table 3: Average Growth Rates (µ) 

Treatment / Dilution

Loading rate

(mg/L)

Average growth rate μ (day^-1) and inhibition of μ (Ir)

0 - 24 hours

0 - 48 hours

0 - 72 hours

µ

Ir (%)

µ

Ir (%)

µ

Ir (%)

Control

-

1.24

0.0

1.50

0.0

1.44

0.0

1:21

4.8

1.24

0.6

1.54

-2.9

1.46

-1.6

1:10

10

1.29

-3.5

1.50

-0.1

1.41

1.5

1:4.6

22

1.28

-3.2

1.46

2.6

1.40

2.2

1:2.1

48

1.07*

13.8

1.44

4.0

1.36*

5.5

Saturated solution

100

1.04*

16.4

1.15*

23.4

0.77*

46.6

*: mean value significantly lower than in the control

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

  

Table 4: Yield (Y) 

Treatment / Dilution

Loading rate

(mg/L)

Yield Y (x 10³) and inhibition of Y (Iy)

0- 2 4 hours

0 - 48 hours

0 - 72 hours

Y

Iy (%)

Y

Iy (%)

Y

Iy (%)

Control

-

2.0

0.0

15.7

0.0

60.0

0.0

1:21

4.8

2.0

1.0

17.2

-9.5

64.6

-7.7

1:10

10

2.1

-5.9

15.7

-0.3

56.0

6.7

1:4.6

22

2.1

-5.8

14.6

6.9

54.7

8.8

1:2.1

48

1.6*

22.4

13.8

11.6

47.1*

21.6

Saturated solution

100

1.5*

25.4

7.4*

53.0

7.3*

87.8

 *: 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

Treatment / Dilution

Loading rate

(mg/L)

Section-by-section growth rates (day^-1) and inhibition of the growth rates (Ir)

0 - 24 hours

24 - 48 hours

48 - 72 hours

µ

Ir (%)

µ

Ir (%)

µ

Ir (%)

Control

-

1.24

0.0

1.76

0.0

1.30

0.0

1:21

4.8

1.24

0.6

1.85

-5.4

1.29

1.5

1:10

10

1.29

-3.5

1.72

2.3

1.24

5.3

1:4.6

22

1.28

-3.2

1.64

6.6

1.29

1.4

1:2.1

48

1.07

13.8

1.81

-3.0

1.19

9.1

Saturated solution

100

1.04

16.4

1.26

28.4

0.00

99.8

 

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

Treatment / Dilution

Loading rate

(mg/L)

Phosphate (mg PO4/L)

0 hours

24 hours

48 hours

72 hours

Sample 1 + 2

mean

Sample 1 + 2

mean

Sample 1 + 2

mean

Sample 1 + 2

mean

Control

-

1.24

1.24

1.24

0.99

1.03

1.01

0.80

0.76

0.78

0.07

0.19

0.13

1:21

4.8

1.12

1.13

1.13

1.11

1.07

1.09

0.75

0.82

0.78

0.13

0.06

0.09

1:10

10

1.09

1.01

1.05

0.98

0.99

0.99

0.75

0.76

0.75

< 0.03

< 0.03

< 0.03

1:4.6

22

0.93

0.93

0.93

0.80

0.88

0.84

0.61

0.58

0.59

< 0.03

< 0.03

< 0.03

1:2.1

48

0.60

0.62

0.61

0.59

0.46

0.53

0.25

0.21

0.23

< 0.03

< 0.03

< 0.03

Saturated solution

100

< 0.03

< 0.03

< 0.03

< 0.03

< 0.03

< 0.03

< 0.03

< 0.03

< 0.03

< 0.03

< 0.03

< 0.03

 

Validity criteria fulfilled:
yes
Remarks:
The control biomass is multiplied by 74 over 72 hours (threshold >,16), the mean coeff. of variation of the daily growth rates was 20% (threshold < 35%), and the coeff. of variation of the average specific growth rates was 2.1% (threshold < 7%).
Conclusions:
The test material had a statistically significant inhibitory effect on the growth (AUC, growth rate and yield) of Scenedesmus subspicatus after the test period of 72 hours at the loading rate of 48 mg/L and above. Thus, this loading rate was determined as the 72 hour LOELR. The 72 hour NOELR was determined to be the loading rate of 22 mg/L. The 72 hour EL50 was > 100 mg/L. However, a significant phosphate depletion was observed at the highest tested concentrations, probably due to a complexation process with the test material. As a consequence, it is possible that the reduction of growth observed was caused by an indirect effect of phosphate lack, rather than a toxic effect of the test material.
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
From 14 December 2007 to 27 February 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 preculture 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
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
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) during stirring of the dispersion. 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 1.01 x 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 exp 3 *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 µ (day-1) and inhibition of µ (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 in the environment) during stirring of the dispersion. 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, the cultures of 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 the OECD Guideline 201 (2006).  The NOEC, the LOEC and EC50 values based on the growth rate were 32 mg/L, 100 mg/L and > 100 mg/L, respectively.   

 

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

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 in the environment) during stirring of the dispersion. 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.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
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:
yes
Remarks:
the phosphate concentration in the test media was analysed, in order to assess potential phosphate depletion.
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:
potasssium 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 72 h period. As the substance could not be detected (< LOQ), the results are based on nominal concentrations. The ErC50 was > 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.
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: 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 metal 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 test 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
Details on results:
ZrOCl2 precipitates phosphate and limits algal growth at 100 ppm at a pH range of 2-11. At this concentration, ZrOCl2 does not seem to be harmful to the algae.
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 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 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 of 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.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
Read across from studies performed with praseodymium(III,IV) oxide, the reaction mass of cerium dioxide and zirconium dioxide, and studies used to cover the endpoint for zirconium dioxide (i.e. next to the latter study, studies performed with zirconium basic carbonate and zirconium dichloride oxide). The read across justification document is attached to 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
Reason / purpose for cross-reference:
read-across source
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: The reaction mass of cerium dioxide, praseodymium(III,IV) oxide and 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 reaction mass of cerium dioxide and zirconium dioxide (Peither, 2009a), praseodymium(III,IV) oxide (Peither, 2009b) and studies used for covering the endpoint for zirconium dioxide (next to the study of Peither (2009a), studies performed with zirconium basic carbonate (Vryenhoef and Mullee, 2010) and zirconium dichloride oxide (Kumar and Rai, 1978)).

Description of key information

The endpoint is covered by a weight of evidence approach including studies performed with the reaction mass of cerium dioxide and zirconium dioxide (Peither, 2009a), praseodymium(III,IV) oxide (Peither, 2009b) and studies used to conclude on the endpoint for zirconium dioxide (next to the study of Peither (2009a), studies performed with zirconium basic carbonate (Vryenhoef and Mullee, 2010) and zirconium dichloride oxide (Kumar and Rai, 1978). All EC50 values were > 100 mg/L (nominal loading rate), consequently, the individual constituents of the reaction mass are not considered as toxic or harmful to algae. This leads to the conclusion that the reaction mass of cerium dioxide, praseodymium(III,IV) oxide and zirconium dioxide is not toxic or harmful to algae either. 

Key value for chemical safety assessment

Additional information

1. Information on zirconium dioxide

For concluding on the toxicity of zirconium dioxide to aquatic algae and cyanobacteria, three studies were included in the weight of evidence approach to cover this endpoint. All three studies were performed with substances other than zirconium dioxide. On the one hand, a study with a 'water soluble' zirconium compound (zirconium dichloride oxide) was included. On the other hand, two studies with insoluble zirconium compounds (zirconium basic carbonate and a reaction mass of zirconium dioxide and cerium dioxide) were included.

The first study (Vryenhoef and Mullee, 2010) investigated the effect of zirconium basic carbonate 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 only considered relevant in limited test systems such as those used for standard aquatic toxicity testing.

In the study by Peither (2009a; according to OECD 201 and GLP), a reaction mass of ca. 60% CeO2 and 30% ZrO2 was tested at loading rates up to 100 mg/L in Scenedesmus subspicatus for 72 hours. 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. The loss of phosphate can be explained by the formation of insoluble complexes of phosphate with the test item (which is a well-known behavior of rare earth elements as well as zirconium in the environment) during stirring of the dispersion. The observed algal growth inhibition was concurrent with the depletion of phosphate in the test medium and therefore the observed effect is considered a secondary effect and not environmentally relevant.

Finally, in the study by Kumar and Rai (1978), 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. In conclusion, zirconium dichloride oxide is not expected to be toxic to algae in the natural aquatic environment. The relation between zirconium dichloride oxide and zirconium dioxide is that in a buffered test medium zirconium dichloride oxide hydrolysis will be completed, resulting in formation of zirconium dioxide which precipitates from solution. Exposing aquatic organisms to 'water soluble' or insoluble zirconium compounds will hence not result in substantially different test results.

Despite the observation of a secondary effect on growth rate due to phosphate deprivation, the EC50 levels obtained from these read across studies would not give rise to classification of zirconium dioxide as harmful to aquatic organisms (all EC50 values > 100 mg/L).

2. Information on reaction mass of cerium dioxide and zirconium dioxide

The results of the guideline study performed by Peither (2009a) are already described above, as they were also used to draw conclusions on the endpoint for zirconium dioxide.

3. Information on praseodymium(III,IV) oxide

In a guideline study (Peither, 2009b), it was observed that praseodymium(III,IV) oxide had a statistically significant inhibitory effect on the growth of Scenedesmus subspicatus after the test period of 72 hours at the loading rate of 48 mg/L and above. Thus, this loading rate was determined as the 72 hour LOELR. The 72 hour NOELR was determined to be the loading rate of 22 mg/L. The 72 hour EL50 was > 100 mg/L. However, a significant phosphate depletion was observed at the highest tested concentrations, probably due to a complexation process with the test material. As a consequence, it is possible that the reduction of growth observed was caused by an indirect effect of phosphate deprivation, rather than a toxic effect of the test material itself.

4. Conclusion on the reaction mass of cerium dioxide, praseodymium(III,IV) oxide and zirconium dioxide

Based on the results explained above obtained for its individual constituents, it can be concluded that the reaction mass of cerium dioxide, praseodymium(III,IV) oxide and zirconium dioxide is not toxic or harmful to algae either.