Magnesium zirconium oxide

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

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

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 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 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 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 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 behavior 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 behavior 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.

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Qualifier:

according to guideline

Guideline:

ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)

Qualifier:

according to guideline

Guideline:

other: OECD series on testing and assessment number 23

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Singular samples for possible analysis were taken from the undiluted filtrate and the control according to the schedule below.
- Frequency at t=0, t=24 and t=72 h. At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.
- Volume: 4.8 mL
- Storage: Samples were stored in a freezer until analysis
- Pre-treatment: On the day of analysis, the frozen samples were defrosted at room temperature. The test samples were diluted with 0.2 mL HNO3 and analysed. If neccesary, the samples were further diluted with 4% (v/v) aqueous HNO3 to obtain concentrations within the calibration range.

Vehicle:

yes

Details on test solutions:

- Preparation of test solutions: The standard test procedures required generation of test solutions, which contained completely dissolved test substance concentrations or stable and homogeneous mixtures or dispersions. The batch of magnesium hydroxide tested was a white powder and the substance appeared not completely soluble in the test medium at the concentrations tested. Preparation of test solutions started with an aqueous dispersion of 100 mg/L applying 3 days of magnetic stirring. The pH of the medium was adjusted from 8.9 to 6.5 using hydrochloric acid before the start of stirring. Magnetic stirring resulted in a dispersion. The dispersion was then filtered through a 0.45 µm membrane filter to remove the undissolved material. The filtrate was used as highest test concentration and the lower concentrations were prepared by subsequent dilution in test medium. The control received similar treatment to prevent any treatment related effect. After preparation, volumes of 50 mL were added to each replicate of the respective test concentration. Subsequently, 1 mL of an algal suspension was added to each replicate providing a cell density of 10^4 cells/mL.
- Test concentrations: magnesium hydroxide: 0.10, 1.0, 10 and 100% of a 45 µm filtered solution prepared at 100 mg/L.
- Controls: Test medium without test substance or other additives.

Test organisms (species):

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

Details on test organisms:

- Common name: Fresh water algae
- Strain: NIVA CHL 1
- Source: In-house laboratory culture
- Reason for selection: This system is an unicellular algal species sensitive to toxic substances in the aquatic ecosystem and has been selected as an internationally accepted test species.

ACCLIMATION
- Stock culture: Algal stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C.
- Culturing media and conditions: 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 10^4 cells/mL. The pre-culture was maintained under the same conditons as used in the test.
- Appearance of the cells: At the end of the final test microscopic observations were performed on the limit concentration to verify a normal and healthy appearance of the inoculum culture and to observe for any abmormal appearance of the algae.
Light intensity: 60 to 120 µE/m^2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

- Temperature of medium: Continuously monitored in a temperature control vessel.
- The temperature of the test medium was 22.9°C at the start of the test. During the exposure period the temperature measured in the incubator was maintained between 21.7 and 24.1°C. Hence, temperature generally remained within the limits prescribed by the protocol (21-24°C).

pH:

- pH was recorded at the beginning and the end of the test.
- At the end of the test the pH was within the limits prescribed by the protocol (6.0-9.0, preferably not varying more than 1.5 unit).
- pH of control: 7.1
- pH of 100 mg/L Mg(OH)2: 6.5
- At the start of the test pH of both was 6.8.

Salinity:

not applicable

Nominal and measured concentrations:

Test Concentrations:
- Magnesium hydroxide: 0.10, 1.0, 10 and 100% of a 0.45 µm filtered solution prepared at 100 mg/L.
- Replicates: 6 replicates of the control and undiluted filtrate, 3 replicates of each dilution of the filtrate, 1 extra replicate of the control and undiluted filtrate, for sampling purposes, 1 or 2 replicates of each test concentration without algae.

Details on test conditions:

Growth Medium:
- Stock Culture Medium: M1, according to the NPR 6505 formulated using Milli-RO water.
- Pre-culture medium: M2, according to the OECD 201 Guideline, formulated using Milli-Q water and subsequently passed over activated carbon and ion-exchange cartridges preventing precipitation.

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:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

- In the controls, cell density increased by an average factor of > 16 within two days - See Table 1.
- Analyses of samples taken from the filtered 100 mg/L solution showed that the measured concentration was in agreement with the nominal throughout the test period (95-96%). Hence, the precipitate that was removed from the solution before the start of the test was not test substance related.
- Growth rates were in the range of the controls at the concentrations from 0.1 to 10 mg/L during the 72-hour test period, whereas the growth rate of algae exposed to 100 mg/L was only slightly reduced (8.2%) (Tables 2 and 3).
- Magnesium hydroxide slightly reduced growth rate and inhibited the yield of the freshwater algal species Pseudokirchneriella subcapitata at 100 mg/L, being the highest concentration. However, a 50% effect level could not be reached.
- The essential endpoint, being the EC50 for growth rate reduction, was determined applying the approaches as recommended in the OECD guidline 201. In addition, the EC50 for yield inhibition was determined as additional parameter.
- No EC50 values could be calculated because the observed effects were lower than 50%.
- The mean coefficient of variation for section-by-section specific growth rates in the control cultures did not exceed 35%.
- No biologically significant inhibition of yield was observed at concentrations between 0.1 and 10 mg/L. Inhibition of yield at 100 mg/L was significant with 31.7%.

Results with reference substance (positive control):

- The EC50 for growth rate reduction (ErC50: 0-72h) was 1.2 mg/L with a 95% confidence interval ranging from 0.77 to 2.0 mg/L. The historical ranges for growth rate reduction lie between 0.82 and 2.3 mg/L. hence, the ErC50: 0-72h for the present batch corresponds with this range.
- The EC50 for yield inhibition (EyC50: 0-72h) was 0.44 mg/L with a 95% confidence interval ranging from 0.37 to 0.54 mg/L. The historical ranges of the 72h EC50 for yield inhibition lie between 0.43 and 1.1 mg/L. Hence, the EyC50: 0-72h for the present batch corresponds with this range.

Table 1 shows mean cell densities measured at 24-hour intervals at the different concentrations of magnesium hydroxide.

Table 1: Mean cell densities (x 10⁴ cells/mL)

Concentration magnesium hydroxide (mg/L)	Exposure time (hours)
	0	24	48	72
Control	1.0	6.6	24.2	103.4
0.10	1.0	6.2	22.6	99.9
1.0	1.0	6.5	23.7	103.7
10	1.0	6.7	23.0	99.9
100	1.0	5.7	19.4	71.0

 

Table 2 shows the calculation of the percentages of growth rate reduction (total test period) and the percentages of yield inhibition.

 

Table 2: Percentage reduction of growth rate (total test period) and percentage inhibition of yield 

Concentration magnesium hydroxide (mg/L)	Mean growth rate		Yield (0-72 h)
	µ (0-72 h)	Reduction (%)	x 10^4cells/mL	Inhibition (%)
Control	0.06440		102.42
0.10	0.06392	0.7	98.92	3.4
1.0	0.06445	-0.1	102.72	-0.3
10	0.06391	0.8	98.93	3.4
100	0.05915	8.2	70.00	31.7

 

 

Table 3 shows the calculation of the percentages of growth rate reduction at different time intervals.

Table 3: Percentage reduction of growth rate at different time intervals

Concentration magnesium hydroxide (mg/L)	Mean growth rate
	µ (0-24 h)	Reduction (%)	µ (24-48 h)	Reduction (%)	µ (48-72 h)	Reduction (%)
Control	0.07833		0.05444		0.06044
0.10	0.07576	3.3	0.05409	0.6	0.06192	-2.4
1.0	0.07818	0.2	0..05357	1.6	0.06159	-1.9
10	0.07929	-1.2	0.05623	-3.3	0.06120	-1.3
100	0.07220	7.8	0.05128	5.8	0.05396	10.7

 

 

 Reference Test:

- Algae were exposed for a period of 72 hours to K2Cr2O7 (Potassium dichromate) concentrations of 0.18, 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L and to a control.

- The initial cell density was 1.0 x 10^4 cells/mL.

- The results are recorded in Table 4.

Table 4: Overview of % reduction of growth rate and % inhibition of yield in the reference test

Nominal Conc. K2Cr2O7   (mg/L)	Mean Growth Rate		Yield (0-72 h)
	µ (0-72 h)	Reduction (%)	x 10^4cells/mL	Inhibition (%)
Control	0.07700		254.78
0.18	0.07620	1.0	240.51	5.6
0.32	0.07274	5.5	187.13	26.6
0.56	0.06275	18.5	90.76	64.4
1.0	0.03971	48.4	16.46	93.5
1.8	0.02597	66.3	5.49	97.8
3.2	0.02011	73.9	3.26	98.7

 

Potassium dichromate reduced growth rate of this fresh water algae at nominal concentrations of 0.56 mg/L and higher.

The EC50 for growth rate reduction (ErC50: 0-72 h) was 1.2 mg/L with a 95% confidence interval ranging from 0.77 to 2.0 mg/L.

The EC50 for yield inhibition (EyC50:0 -72 h) was 0.44 mg/L with a 95% confidence interval ranging from 0.37 to 0.54 mg/L.

 

 

 

Conclusions:

Under the conditions of the present study with Pseudokirchneriella subcapitata, magnesium hydroxide slightly reduced growth rate and inhibited the yield of this fresh water algae species at 100 mg/L, being the highest test concentration. However, a 50% effect level could not be reached. In conclusion: both the EC50 for growth rate reduction (ErC50: 0-72 h) and the EC50 for yield inhibition (EyC50: 0-72 h) exceeded an analytically confirmed concentration of 100 mg/L.

Reference 3

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.

Reference 4

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.

Reference 5

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 the zirconium compounds zirconium basic carbonate, zirconium dichloride oxide, and reaction mass of cerium dioxide and zirconium dioxide, and a study performed with magnesium dihydroxide. The read across justification document is attached in IUCLID Section 13.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Key result

Remarks on result:

other: Magnesium zirconium oxide is not considered to be toxic or harmful to aquatic algae.

Remarks:

This conclusion was based on the results of the studies from Vryenhoef and Mullee (2010) with zirconium basic carbonate, Kumar and Rai (1978) with zirconium dichloride oxide, Peither (2009) with a reaction mass of cerium dioxide and zirconium dioxide, and Migchielsen (2010) with magnesium dihydroxide.

Description of key information

The endpoint is covered by a weight of evidence approach including three studies for zirconium compounds (Vryenhoef and Mullee, 2010; Peither, 2009; Kumar and Rai, 1978) and one study for magnesium hydroxide (Migchielsen, 2010). Data on magnesium hydroxide were included in the dossier because magnesium oxide is transformed to magnesium hydroxide when in contact with water. All EC50 values were > 100 mg/L, consequently, the individual compounds magnesium oxide/magnesium hydroxide and zirconium dioxide are not considered as harmful to algae. This leads to the conclusion that magnesium zirconium oxide is not expected to be harmful to algae either. 

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 magnesium oxide

For magnesium oxide, data obtained from a test performed with magnesium hydroxide were added to the dossier. The rationale behind this is that in the environment, MgO will result in Mg(OH)2 formation when in contact with water, according to the following (general) reaction:

MgO + H2O <--> Mg(OH)2

The study under consideration (Migchielsen, 2010) investigated the effect of magnesium hydroxide on the growth rate of Pseudokirchneriella subcapitata. The 72-h ErC50 exceeded an analytically confirmed concentration of 100 mg/L and would not give rise to classification of the substance as harmful to aquatic organisms (EC50 > 100 mg/L).

3. Conclusion on magnesium zirconium oxide

The adverse effects of zirconium compounds in algae were concluded to be caused by phosphate depletion, whereas no adverse effects were observed with Mg(OH)2 (relevant for MgO). The effect with the zirconium compounds was considered to be a secondary effect. The EC50 values obtained from the studies included in the weight of evidence approach would not give rise to classification of zirconium dioxide or magnesium oxide/hydroxide as harmful to aquatic organisms (all EC50 values > 100 mg/L). Consequently, it can be concluded that magnesium zirconium oxide should not be considered as harmful to algae either.