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Ecotoxicological information

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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
No analytic were performed. No international guideline was used for the present study. Little information regarding the material and methods used. Read across to ZrOCl2 is used as solutions of ZrOCl2 adjusted to neutral pH contain high amounts of ZrO2 due to the hydrolysis reaction of ZrOCl2.
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 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:
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 Chl. 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, growth inhibition observed, was 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:
experimental study
Adequacy of study:
weight of evidence
Study period:
From 14-DEC-2007 to 15-MAY-2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was performed according to an international test guideline and to GLP.
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: triplicates 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 analyzed, 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 stabilize 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 equilibrations 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:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (total fraction)
Basis for effect:
growth rate
Remarks on result:
other: 95% CI not determined
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
32 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (total fraction)
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOELR
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (total fraction)
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. (dissolved fraction)
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. (dissolved fraction)
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
42 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (dissolved fraction)
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 behavior 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 ¿g/L at the loading rates of 3.2 to 32 mg/L. At the highest loading rate of 100 mg/L, the concentration of cerium were 27, 29, 19 and 4 ¿g/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 EL10 and EL50 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 EL90 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 LOELR and NOELR, 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 to1.01x 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 72h-LOELR. The 72h-EL50 was > 100 mg/L. The NOELR 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 behavior 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.
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 NOELR , the LOELR and EL50 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 behavior 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.

Description of key information

In view of the lack of algae test data on ZrO2, read across is proposed to ZrOCl2 and to reaction mass of ZrO2 and CeO2.
Both studies show phosphate depletion due to the presence of zirconium compounds in the medium, which causes algae growth inhibition. No direct toxic effects caused by ZrO2 are to be expected.

Key value for chemical safety assessment

Additional information

In the study by Peither (2009; 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 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.

In the Kumar and Rai study (1979), a similar effect was observed when exposing Chlorella vulgaris to different concentrations of ZrOCl2. ZrOCl2 when in water hydrolyses into the insoluble ZrO2, especially when the pH is raised to neutral. Phosphate precipitation was observed in the study, which caused growth inhibition of the algae. It was emphasized that ZrOCl2 (or ZrO2) is not directly toxic to algae, but that the growth inhibition is caused by phosphate depletion.