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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:
22 November 2012 - 27 November 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
A sample of each test concentration was taken for chemical analysis at 0 and 72 hours in order to determine the stability of the test item under test conditions. All samples were stored frozen prior to analysis.
The test samples were thawed with the aid of sonication. Nitric acid (4 mL) was added to a volume (200 mL) of sample and the samples were sonicated for 15 minutes before being filtered through 0.45 µm cellulose acetate filters.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
Method: Due to the low aqueous solubility of the test item, a dispersion was prepared by adding 100 mg of test item to the surface of 2 liters of culture medium and stirred vigorously using a magnetic stirrer for 10 minutes after which the pH was adjusted from 6.3 to 7.5. The media was then stirred for a further 24 hours. After stirring, any undissolved test item was removed by filtration through a 0.2 µm Gelman Acrocap filter (initial 500 mL discarded) to give a 100% v/v saturated stock solution. The test item used represents a solution containing 29.2% anhydrous zirconium dichloride oxide. Given the low aqueous solubility it was considered that a loading rate of 50 mg test item/L, which corresponds to 14.6 mg zirconium dichloride oxide/L, was sufficient to ensure 100% saturation of the test media. A series of dilutions was made from the 100% v/v saturated solution to give further stock solutions of 10, 1.0 and 0. 10% v/v saturated solution. An aliquot (450 mL) of each of the stock solutions was separately inoculated with algal suspension (3.9 mL).
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata strain
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland.
- Method of cultivation: Prior to the start of the test sufficient master culture was added to approximately 100 mL volumes of culture media contained in conical flasks to give an initial cell density of approximately 1000 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100–150 rpm) and constant illumination at 24 ± 1 °C until the algal cell density was approximately 10,000-100,000 cells/mL.

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


Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
no data
Test temperature:
24± 1°C
pH:
Control:
0 h: 7.7
72 h: 8.0
Treatments:
0 h: 7.5-7.7
72 h: 7.8-8.0
Dissolved oxygen:
not applicable
Salinity:
not applicable
Nominal and measured concentrations:
Nominal (% v/v saturated solution): 0, 1.0, 10, 100
Measured: 100% v/v saturated solution: < 0.011 mg Zr/L (LOQ)
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL conical flasks
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 250 mL glass conical flasks each containing 100 mL of test preparation and plugged with polyurethane foam bungs to reduce evaporation
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2

GROWTH MEDIUM
- Standard medium used: yes

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

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

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Range finding study: yes
- Test concentrations: 0.10, 1.0, 10 and 100% v/v saturated solution.
- Results used to determine the conditions for the definitive study: Chemical analysis of the 10 and 100% v/v saturated solution test preparations at 0 and 72 hours showed that dissolved zirconium concentrations were less than the limit of quantitation, which was determined to be 0.011 mg Zr/L. As no zirconium was in solution, it was considered unnecessary to continue to the definitive stage of testing.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
80 other: % v/v saturated solution
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
10 other: % v/v saturated solution
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no abnormalities observed
- Any stimulation of growth found in any treatment: no
- Observed effects: In the 100% v/v saturated solution, 73% inhibition (growth rate-based) was observed after 72 h. The EC50 and NOEC based on growth rate were reported to be 80% and 10% v/v saturated solution, respectively. However, dissolved zirconium appeared to be < LOQ (i.e. 11 µg Zr/L) in all of the treatments, indicating that the observed inhibition of growth may be due to a secondary effect.
Results with reference substance (positive control):
ErC50 (0 – 72 h): 1.1 mg/L; 95% confidence limits 1.0 – 1.3 mg/L
EbC50 (0 – 72 h): 0.70 mg/L
The results from the positive control with potassium dichromate were within the normal ranges for this reference item.
Reported statistics and error estimates:
For each test concentration % inhibition was plotted against test concentration (the latter on a logarithmic axis) and a line fitted by computerized interpolation using the Xlfit software package (IDBS). ECx values were then determined from the equation for the fitted line.
The NOEC was determined by visual inspection of the growth rate and yield data.
Validity criteria fulfilled:
yes
Conclusions:
In this study, it was impossible to keep zirconium dissolved at measurable levels under the conditions of the test (i.e., dissolved Zr < LOQ = 11 µg Zr/L). However, after 72 h of exposure of Pseudokirchneriella subcapitata to a 100% v/v saturated solution, significant growth inhibition was observed, resulting in a 72-h ErC50 and NOErC of 80 and 10% v/v saturated solution. Because no bioavailable (dissolved) zirconium was detected in the test media, the observed toxicity may be rather due to phosphate deprivation (see other studies) which is a secondary effect and not considered environmentally relevant.
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 basic carbonate and a reaction mass of cerium dioxide and zirconium dioxide. The read across justification document is attached in IUCLID Section 13.
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
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: Zirconium dichloride oxide is not considered to be toxic or harmful to aquatic algae.
Remarks:
This conclusion was based on the results of studies performed with the read across substances zirconium 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).

Description of key information

A weight of evidence approach was performed using four studies, two studies with zirconium dichloride oxide, which is a 'water soluble' zirconium compound (Vryenhoef, 2014; Kumar and Rai, 1978), one with zirconium basic carbonate, which is a sparingly soluble zirconium compound (Vryenhoef and Mullee, 2010), and one with a reaction mass of cerium dioxide and zirconium dioxide, which is an insoluble zirconium compound (Peither, 2009). The latter two studies report an adverse effect on algal growth which is concurrent with phosphate depletion from the test medium through complexation and precipitation with zirconium. Although zirconium dichloride oxide is a 'water soluble' zirconium compound, the similarity of its hazard profile with the profiles of insoluble or sparingly soluble zirconium compounds is very high, because zirconium precipitates from the solution at environmentally relevant conditions, as was demonstrated by zirconium analysis in algal test media (in all samples, dissolved Zr was < LOQ, i.e. < 11 µg Zr/L, Harris, 2014). Therefore, read across from insoluble or sparingly soluble zirconium compounds is considered acceptable. Further, although phosphate monitoring was not performed in the study with zirconium dichloride oxide conducted by Harris (2014), a similar phosphate depletion effect can be expected to have occurred, because zirconium is known to heavily complex all phosphates (independent of pH) whenever it is in excess of the phosphates (and vice versa). This is confirmed by the results from the study of Kumar and Rai (1978). Further, pH-dependent formation of zirconium dioxide, zirconium hydroxide, and zirconium carbonate precipitates can be expected in environmentally relevant test media. Based on the weight of evidence approach, the observed effects on algae are considered secondary effects which are not environmentally relevant. In conclusion, the test substance is not considered to be toxic to algae.

Key value for chemical safety assessment

Additional information

For toxicity to aquatic algae and cyanobacteria, four studies were included in this dossier and used in a weight of evidence approach to cover this endpoint.

The most recent test was performed using zirconium dichloride oxide (Vryenhoef, 2014). A range finding experiment indicated that no measurable zirconium could stay in solution (i.e., dissolved Zr < LOQ, i.e. < 11 µg Zr/L). In the 100% v/v saturated solution, significant growth inhibition was however observed. The 72-h EC50 and NOEC based on growth rate were 80 and 10% v/v saturated solution, respectively. Because of the absence of bioavailable (dissolved) zirconium in the test media, no further testing was deemed necessary and the observed effect was considered to be a secondary effect (i.e., growth inhibition due to phosphate deprivation). Because at environmentally relevant conditions, zirconium precipitates out of solution (predominantly as zirconium hydroxide, zirconium dioxide, zirconium phosphate, and zirconium carbonate), read across from insoluble and/or sparingly soluble zirconium substances is considered acceptable.

The second study performed with zirconium dichloride oxide is the study by Kumar and Rai (1978), in which it is shown that algae exposed to zirconium dichloride oxide up to 100 ppm show growth inhibition, especially at 60, 80 and 100 ppm. This effect is caused by precipitation of phosphates which are essential to algae. When algae are supplemented with phosphate in the medium after filtration, growth was comparable to controls. The results suggest that zirconium dichloride oxide is not toxic directly to algae at concentrations up to 100 ppm. In conclusion, zirconium dichloride oxide is not expected to be toxic to algae in the natural aquatic environment.

To provide further evidence on the phosphate deprivation effect typically observed for zirconium substances, two read across studies were added to the weight of evidence approach. The first study (Vryenhoef and Mullee, 2010) investigated the effect of zirconium basic carbonate (a sparingly soluble zirconium compound) on the growth of Desmodesmus subspicatus over a 72 h period. As zirconium could not be detected (< LOQ) in the test solution, the results were based on nominal concentrations. The ErC50 was > 100 mg/L and the NOErC was 32 mg/L (based on zirconium basic carbonate). Phosphate monitoring during the test indicated that reduced growth rate was concurrent with phosphate depletion due to phosphate complexing with zirconium and precipitation of the formed complexes. The observed effect is clearly a secondary effect which is not considered environmentally relevant.

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