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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:
2018-01-12 to 2018-06-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Sampling method: Two samples were taken from the control and each test solution at 0 hours and from the pooled replicates at 72 hours for analysis of dissolved zirconium and tungsten.
- Sample storage conditions before analysis: The 0-h test samples were processed on the day of receipt, stored refrigerated, and then analysed by ICP-MS analysis alongside the 72-h test samples.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
*Stirring experiment:
A nominal amount of test item (500 mg) was dispersed in 5 liters of culture medium with the aid of vigorous magnetic stirring for 96 hours. After 3, 24 and 96 hours, the pH of the media was checked and a sample taken for chemical analysis (i.e. dissolved tungsten) after filtration through a 0.2 µm Gelman Acrocap filter (initial 100 mL discarded). Dissolved tungsten concentrations were 0.115, 0.109 and 0.125 mg W/L after 3, 24 and 96 hours of stirring, respectively. Based on these results, the 3-h stirring period was selected for the definitive test.

*Definitive test:
A nominal amount of test item (500 mg) was dispersed in 5 liters of culture medium with the aid of vigorous magnetic stirring for 3 hours. After 3 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 µm Gelman Acrocap filter (first approximate 100 mL discarded in order to precondition the filter) to give a 100% v/v saturated solution. A series of dilutions was made from this saturated solution to give stock solutions of 50, 25, 12.5 and 6.25% v/v saturated solution. An aliquot (500 mL) of each of the stock solutions was separately inoculated with 1.3 mL of algal suspension to give the required test concentrations of 6.25, 12.5, 25, 50 and 100% v/v saturated solution. The stock solutions and each prepared concentration were inverted several times to ensure adequate mixing and homogeneity.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Liquid cultures of Pseudokirchneriella subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland.
- Method of cultivation: Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were kept under constant agitation by orbital shaker (approximately 150 rpm) and constant illumination at 24 ± 1°C.
- 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 10^3 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (approximately 150 rpm) and constant illumination at 24 ± 1°C until the algal cell density was approximately 10^4 to 10^5 cells/mL. These pre-cultures were used for inoculation of the test solutions at an initial cell density of 5E+03 cells/mL.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
14.8 mg CaCO3/L
Test temperature:
24 ± °1C
pH:
t=0h: 7.5-7.6
t=72h: 7.8-8.3
Dissolved oxygen:
not applicable
Salinity:
not applicable
Nominal and measured concentrations:
Nominal concentrations: 0, 6.25, 12.5, 25, 50 and 100% v/v Saturated Solution (nominal loading rate 100 mg/L)
Dissolved zirconium concentrations were < LOQ in all test media at all sampling occasions.
Dissolved tungsten concentrations (mg W/L):
*t=0h: < LOQ, 0.00564, 0.0114, 0.0220, 0.0411, 0.0763
*t=72h: < LOQ, 0.00695, 0.0126, 0.0216, 0.0445, 0.0925
Details on test conditions:
TEST SYSTEM
- Test vessel: 250-mL glass conical flasks
- Type (delete if not applicable): plugged with polyurethane foam bungs
- Material, size, headspace, fill volume: 250-mL glass conical flasks filled with 100 mL test solution
- Initial cells density: 5,000 cells/mL
- Control end cells density: 1.32 x 10^6 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes
* NaNO: 325.5 mg/L
* MgCl2.6H2O: 12.16 mg/L
* CaCl2.2H2O: 4.41 mg/L
* MgSO4.7H2O: 14.6 mg/L
* K2HPO4: 1.044 mg/L
* NaHCO3: 15.0 mg/L
* H3BO3: 0.186 mg/L
* MnCl2.4H2O: 0.415 mg/L
* ZnCl2: 0.00327 mg/L
* FeCl3.6H2O: 0.160 mg/L
* CoCl2.6H2O: 0.00143 mg/L
* Na2MoO4.2H2O: 0.00726 mg/L
* CuCl2.2H2O: 0.000012 mg/L
* Na2EDTA.2H2O: 0.30 mg/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The culture medium was prepared using reverse osmosis purified deionised water.
- Culture medium different from test medium: no
- Intervals of water quality measurement: The pH of the control and each test preparation was determined at initiation of the test and after 72 hours of exposure. The temperature within the incubator was recorded daily. The appearance of the test media was recorded daily.

OTHER TEST CONDITIONS
- Sterile test conditions: no information
- Adjustment of pH: yes, to 7.5 with 0.1 N NaOH or HCl
- Photoperiod: continuous illumination
- Light intensity and quality: intensity approximately 7000 lux provided by warm white lighting (380 to 730 nm)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Determination of cell concentrations [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter]: At the start of the test and after 72 h a sample of each test and control culture was removed and the cell density determined using a Coulter® Multisizer Particle Counter.
- Calculated effect parameters: specific growth rate and yield

TEST CONCENTRATIONS
- Spacing factor for test concentrations: x2
- Range finding study: yes
- Test concentrations in range finding study: 0.1, 1.0, 10 and 100% of the SS
- Results used to determine the conditions for the definitive study: yes (no significant growth inhibition was observed during the range finding study)
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
98 other: % SS with nominal loading rate 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
50 other: % SS with nominal loading rate 100 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
- Any stimulation of growth found in any treatment: no
- Growth inhibition compared to the control was 59% in the 100% saturated solution.
- 72-h EC10 (growth rate) = 87% SS
- 72-h EC20 (growth rate) = 91% SS
- 72-h LOEC (growth rate) = 100% SS
- 72-h EC10 (yield) = 80% SS
- 72-h EC20 (yield) = 83% SS
- 72-h EC50 (yield) = 88% SS
- 72-h NOEC (yield) = 50% SS
- 72-h LOEC (yield) = 100% SS
Results with reference substance (positive control):
- Results with reference substance valid? yes
- 72-h EC50 (growth rate) = 1.6 mg/L (95% C.L. = 1.4 to 1.8 mg/L)
- 72-h EC50 (yield) = 0.77 mg/L (95% C.L. = 0.68 to 0.87 mg/L)
Reported statistics and error estimates:
One way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955) was carried out on the growth rate and yield data after 72 hours for the control, 6.25, 25, 50 and 100% v/v saturated solution test concentrations to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS, 1999 - 2001). The 12.5% v/v saturated solution test concentration was not included in the analysis as visual inspection of the data showed a significant effect on growth.

For each individual test vessel (mean values for yield), percentage inhibition (arithmetic axis) was plotted against test concentration (logarithmic axis) and a line fitted by computerised interpolation using the Xlfit software package (IDBS). The 12.5% v/v saturated solution test group was identified as an outlier and hence disregarded when fitting the concentration response curve. ECx values were then determined from the equation for the fitted line.
Validity criteria fulfilled:
yes
Conclusions:
A 72-h growth inhibition test with the unicellular green alga Pseudokirchneriella subcapitata was performed with the test substance tungsten zirconium hydroxide oxide according to OECD guideline 201 (GLP). The algae were exposed to 6.25, 12.5, 25, 50 and 100% v/v dilutions of a saturated solution with a nominal loading rate of 100 mg/L. Only in the 100% v/v saturated solution, significant growth inhibition was observed in the definitive test (although this was not the case in the range finding study). The growth rate-based 72-h EC50 was determined to be 98% v/v of the SS, whereas the growth rate-based 72-h NOEC was 50% v/v of the SS. Zirconium compounds have been recorded to complex with phosphate by means of an ion exchange mechanism (Soltysik et al., 2016). Phosphate is an important component of the growth media for algae. Therefore, the possibility exists that the effects observed may have been due to the formation of such a complex between the test item and the phosphate in the media, which may have therefore had an indirect impact on the overall growth of the algae in this study. All dissolved zirconium concentrations were < LOQ throughout the test, whereas low concentrations of dissolved tungsten have been measured (up to 0.0905 mg W/L in the 100% v/v saturated solution after 72 h). Unfortunately, no phosphate measurements were performed during the test.
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:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
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:
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 behavior 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 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 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 behavior 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 behavior of rare earth elements 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.

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:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
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.
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:
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.

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 zirconium dichloride oxide, zirconium basic carbonate, and a reaction mass of cerium dioxide and zirconium dioxide. The read across justification document is attached to IUCLID Section 13.
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Remarks on result:
other: Based on the studies performed with zirconium dichloride oxide, zirconium basic carbonate, and the reaction mass of cerium dioxide and zirconium dioxide, zirconium dioxide was considered not to be toxic or harmful to aquatic algae.
Remarks:
These studies performed with the read across substances zirconium dichloride oxide (which is a 'water soluble' zirconium compound) (Kumar and Rai, 1978), zirconium basic carbonate (which is a sparingly soluble zirconium compound) (Vryenhoef and Mullee, 2010), and a reaction mass of cerium dioxide and zirconium dioxide (which is an insoluble zirconium compound) (Peither, 2009) provide evidence for phosphate depletion of the test media when zirconium is in excess, resulting in indirect inhibitory effects in the algae. Because during the study performed with tungsten zirconium (hydroxide) oxide (Vryenhoef, 2018) no phosphate measurements were performed, abovementioned read across studies are included in a weight of evidence approach to be able to nuance the observed effects of tungsten zirconium (hydroxide) oxide in the 100% v/v saturated solution with nominal loading rate of 100 mg/L, resulting in a growth rate-based 72-h EC50 of 98% v/v of the saturated solution.

Description of key information

A weight of evidence approach was performed using four studies: one study with the read across substance zirconium dichloride oxide, which is a 'water soluble' zirconium compound (Kumar and Rai, 1978), one with zirconium basic carbonate, which is a sparingly soluble zirconium compound (Vryenhoef and Mullee, 2010), one with a reaction mass of cerium dioxide and zirconium dioxide, which is an insoluble zirconium compound (Peither, 2009) and a more recent study from Vryenhoef (2018) with the substance tungsten zirconium (hydroxide) oxide.

Based on this weight of evidence approach, the observed effects on algae are considered secondary effects (phosphate deprivation of the algae due to precipitation of zirconium phosphate complexes) which are not environmentally relevant. In conclusion, the test substance is not considered to be harmful or toxic to algae.

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 (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 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 oxide 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 tungsten zirconium (hydroxide) oxide

A 72-h growth inhibition test with the unicellular green alga Pseudokirchneriella subcapitata was performed with the test substance tungsten zirconium hydroxide oxide according to OECD guideline 201 (GLP). The algae were exposed to 6.25, 12.5, 25, 50 and 100% v/v dilutions of a saturated solution with a nominal loading rate of 100 mg/L. Only in the 100% v/v saturated solution, significant growth inhibition was observed in the definitive test (although this was not the case in the range finding study). The growth rate-based 72-h EC50 was determined to be 98% v/v of the SS, whereas the growth rate-based 72-h NOEC was 50% v/v of the SS. All dissolved zirconium concentrations were < LOQ throughout the test, whereas low concentrations of dissolved tungsten have been measured (up to 0.0905 mg W/L in the 100% v/v saturated solution after 72 h). Unfortunately, no phosphate measurements were performed during the test.

3. Conclusion on tungsten zirconium oxide

The growth inhibition observed in the 100% v/v saturated solution with a nominal loading rate of 100 mg/L in the study performed with tungsten zirconium (hydroxide) oxide was most likely an indirect effect resulting from phosphate depletion of the test medium due to precipitation of insoluble zirconium phosphate complexes. Because no phosphate measurements were performed in this study, the read across studies mentioned above (performed with zirconium dichloride oxide, zirconium basic carbonate and a reaction mass of cerium dioxide and zirconium dioxide), which provide evidence for this assumption, are included in a weight of evidence approach together with the study performed with tungsten zirconium (hydroxide) oxide to conclude on the endpoint. Based on this weight of evidence approach, it can be concluded that tungsten zirconium oxide is not harmful or toxic to aquatic algae.