Zirconium dinitrate 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:

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 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

Remarks on result:

other: Zirconium dinitrate 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 dichloride oxide (which is a 'water soluble' zirconium compound) (Vryenhoef, 2014; 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), next to the results of a study performed with zirconium dinitrate oxide itself (Martins et al., 2007).

Reference 2

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: 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.

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:

no

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:

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.

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:

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.

Reference 5

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 analytical monitoring was performed.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline followed

Principles of method if other than guideline:

Two species of green algae, Pseudokirchnerella subcapitata and Pandorina morum, were exposed for 96 h to zirconium dinitrate oxide concentrations ranging from 0.5 to 10 mg Zr/L. Experiments were repeated at four different pH values: 6.5, 7.2, 7.7 and 8. At the end of the 96-hour period, three biomass parameters were evaluated: optical density at 440 nm, cell counting and chlorophyll a concentration. The relationship between the measured endpoints (optical density at 440 nm, cell counting and chlorophyll a concentration) and growth rate was not investigated (despite this is recommended in the current OECD guideline because growth rate-based effect concentrations are considered to be of more ecological relevance than effect concentrations based on biomass-related endpoints).

GLP compliance:

not specified

Analytical monitoring:

no

Details on sampling:

No samples taken for analytical verification of test concentrations.

Vehicle:

no

Details on test solutions:

- Method: ZrO(NO3)2.xH2O was diluted in MBL nutritive culture medium (MBL = Marine Biological Laboratory) to obtain the following test concentrations: 0.5, 2, 4, 6, 8 and 10 mg Zr/L.
- Controls: MBL medium without ZrO(NO3)2.xH2O.

Test organisms (species):

other: Pandorina morum

Details on test organisms:

TEST ORGANISM
- Strain: Müller Bory
- Source (laboratory, culture collection): environmentally obtained and isolated by micromanipulation in the laboratory with a micropipette and a light-microscope
- For inoculation, a culture was incubated for 3-4 days before starting the test under the same conditions as the test cultures - cells were in exponential growing phase.

ACCLIMATION
- Acclimation period: see above, 3-4 days before start of testing / after isolation at least one month cultured in MBL medium in a cabinet (F10 000 EDTU model) and maintained in aseptic conditions
- Culturing media and conditions (same as test or not): same as test

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Post exposure observation period:

none

Hardness:

ca. 40 mg CaCO3/L

Test temperature:

not reported

pH:

test performed at pH 6.5, 7.2, 7.5 and 8.0

Dissolved oxygen:

not applicable

Salinity:

not applicable

Nominal and measured concentrations:

Nominal: Control + 0.5-2.0-4.0-6.0-8.0-10.0 mg Zr/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 100-mL Erlenmeyer flasks, 40-mL fill volume
- Initial cells density: 5x10^4 cells/mL
- Control end cells density: not reported
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
- Medium used: Culture medium from the Marine Biological Laboratory (MBL), composition cfr. typical standard test media

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: sterile distilled water
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: adjustment to envisaged pH using HNO3
- Photoperiod: continuous illumination
- Light intensity and quality: not reported

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): 3 biomass parameters evaluated after 96 h:
- Determination of cell concentrations: cell counting using a Sedgwick-Rafter chamber
- Chlorophyll a measurement: filtration (ca. 20 mL) through Whatman GF/C filters, extraction with acetone (90%), chl a measurement at 665 and 750 nm before and after acidification with HCl (0.1 M).
- Optical density: 6505 UV/VIS spectrophotometer (JENWAY), 440 nm

TEST CONCENTRATIONS
- Range finding study: yes, results not shown
- Results used to determine the conditions for the definitive study: yes
- During preliminary test, a test was also performed to investigate the effect of nitrates. Nitrates at the levels added during the tests with zirconium oxynitrate did not affect algal growth.

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

4.6 mg/L

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

biomass

Remarks on result:

other: at pH 6.5 (95% CI = 3.2-6.5 mg Zr/L)

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

8.1 mg/L

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

biomass

Remarks on result:

other: at pH 7.2 (95% CI = 6.9-9.8 mg Zr/L)

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

biomass

Remarks on result:

other: at pH 7.5 (95% CI = 1.2-5.4 mg Zr/L)

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

2.9 mg/L

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

biomass

Remarks on result:

other: at pH 8.0 (95% CI = 1.2-4.9 mg Zr/L)

Details on results:

In the publication, it is not clear which was the biomass parameter used to calculate the EC50 values. Therefore, in the above table, it was decided to select the generic term of biomass as a basis for effect.

NOEC and LOEC values are not available in the publication, but can be deduced from the data on chlorophyll a measurements:
pH 6.5: NOEC = 0.5 mg Zr/L, LOEC = 2.0 mg Zr/L
pH 7.2: NOEC = 8.0 mg Zr/L, LOEC = 10.0 mg Zr/L
pH 7.5: NOEC = 0.5 mg Zr/L, LOEC = 2.0 mg Zr/L
pH 8.0: NOEC = 0.5 mg Zr/L, LOEC = 2.0 mg Zr/L
The results relative to optical density measurements are not reported in the publication for P. morum.

Reported statistics and error estimates:

EC50 values calculated using Probit analysis. Significant differences between treatments (allowing to deduce NOEC/LOEC values) determined using ANOVA and Tukey's t test for multiple comparison.

Conclusions:

A series of 96-h growth inhibition tests with the green alga Pandorina morum using the test substance zirconium oxynitrate yielded 96-h EC50 values (biomass-based) of 4.6, 8.1, 3.2 and 2.9 mg Zr/L at pH 6.5, 7.2, 7.5 and 8.0, respectively, indicating a slight increase in toxicity with increasing pH. The NOEC values were 0.5, 8.0, 0.5 and 0.5 mg Zr/L, respectively. These NOEC values were deduced from the chlorophyll a measurements reported in the publication, i.e., the NOEC values were biomass-based too. Biomass-based effect concentrations are usually somewhat lower than growth rate-based effect concentrations. It should be kept in mind that growth rate-based effects are considered more ecologically relevant. Finally, in this study, it was also demonstrated that nitrate levels equal to those added during the zirconium oxynitrate assays did not affect algal growth rate. The observed effects can hence be ascribed to the molecule zirconium oxynitrate as such, to the element Zr, or to secondary effects (based on the well-known behavior of complexation with phosphates, see the other endpoint study records).

Reference 6

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 analytical monitoring was performed.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline available

Principles of method if other than guideline:

Two species of green algae, Pseudokirchnerella subcapitata and Pandorina morum, were exposed for 96 h to zirconium dinitrate oxide concentrations ranging from 0.5 to 10 mg Zr/L. Experiments were repeated at four different pH values: 6.5, 7.2, 7.7 and 8. At the end of the 96-hour period, three biomass parameters were evaluated: optical density at 440 nm, cell counting and chlorophyll a concentration. The relationship between the measured endpoints (optical density at 440 nm, cell counting and chlorophyll a concentration) and growth rate was not investigated (despite this is recommended in the current OECD guideline because growth rate-based effect concentrations are considered to be of more ecological relevance than effect concentrations based on biomass-related endpoints).

GLP compliance:

not specified

Analytical monitoring:

no

Details on sampling:

No samples taken for analytical verification of test concentrations.

Vehicle:

no

Details on test solutions:

- Method: ZrO(NO3)2.xH2O was diluted in MBL nutritive culture medium (MBL = Marine Biological Laboratory) to obtain the following test concentrations: 0.5, 2, 4, 6, 8 and 10 mg Zr/L.
- Controls: MBL medium without ZrO(NO3)2.xH2O.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: Korshikov Hindak
- Source (laboratory, culture collection): Alga-Gro Freshwater, Carolina Biological Supply Company, Burlington, North Carolina
- For inoculation, a culture was incubated for 3-4 days before starting the test under the same conditions as the test cultures - cells were in exponential growing phase.

ACCLIMATION
- Acclimation period: see above, 3-4 days before start of testing
- Culturing media and conditions (same as test or not): same as test

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Post exposure observation period:

none

Hardness:

ca. 40 mg CaCO3/L

Test temperature:

not reported

pH:

test performed at pH 6.5, 7.2, 7.5 and 8.0

Dissolved oxygen:

not applicable

Salinity:

not applicable

Nominal and measured concentrations:

Nominal: Control + 0.5-2.0-4.0-6.0-8.0-10.0 mg Zr/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 100-mL Erlenmeyer flasks, 40-mL fill volume
- Initial cells density: 5x10^4 cells/mL
- Control end cells density: not reported
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
- Medium used: Culture medium from the Marine Biological Laboratory (MBL), composition cfr. typical standard test media

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: sterile distilled water
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: adjustment to envisaged pH using HNO3
- Photoperiod: continuous illumination
- Light intensity and quality: not reported

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): 3 biomass parameters evaluated after 96 h:
- Determination of cell concentrations: cell counting using a Newbauer chamber
- Chlorophyll a measurement: filtration (ca. 20 mL) through Whatman GF/C filters, extraction with acetone (90%), chl a measurement at 665 and 750 nm before and after acidification with HCl (0.1 M).
- Optical density: 6505 UV/VIS spectrophotometer (JENWAY), 440 nm

TEST CONCENTRATIONS
- Range finding study: yes, results not shown
- Results used to determine the conditions for the definitive study: yes
- During preliminary test, a test was also performed to investigate the effect of nitrates. Nitrates at the levels added during the tests with zirconium oxynitrate did not affect algal growth.

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

9.8 mg/L

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

biomass

Remarks on result:

other: at pH 6.5 (95% CI = 8.4-12.7 mg Zr/L)

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

7.9 mg/L

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

biomass

Remarks on result:

other: at pH 7.2 (95% CI = 7.1-9.1 mg Zr/L)

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

7.3 mg/L

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

biomass

Remarks on result:

other: at pH 7.5 (95% CI = 6.3-8.6 mg Zr/L)

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

6.2 mg/L

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

biomass

Remarks on result:

other: at pH 8.0 (95% CI = 5.4-7.3 mg Zr/L)

Details on results:

In the publication, it is not clear which was the biomass parameter used to calculate the EC50 values. Therefore, in the above table, it was decided to select the generic term of biomass as a basis for effect.

NOEC and LOEC values are not available in the publication, but can be deduced from the data on optical density measurements:

pH 6.5: NOEC = 4.0 mg Zr/L, LOEC = 6.0 mg Zr/L
pH 7.2: NOEC = 4.0 mg Zr/L, LOEC = 6.0 mg Zr/L
pH 7.5: NOEC = 2.0 mg Zr/L, LOEC = 4.0 mg Zr/L
pH 8.0: NOEC = 2.0 mg Zr/L, LOEC = 4.0 mg Zr/L
The results relative to chlorophyll a are not reported in the publication for P. subcapitata.

Reported statistics and error estimates:

EC50 values calculated using Probit analysis. Significant differences between treatments (allowing to deduce NOEC/LOEC values) determined using ANOVA and Tukey's t test for multiple comparison.

Conclusions:

A series of 96-h growth inhibition tests with the unicellular green alga Pseudokirchneriella subcapitata using the test substance zirconium oxynitrate yielded 96-h EC50 values (biomass-based) of 9.8, 7.9, 7.3 and 6.2 mg Zr/L at pH 6.5, 7.2, 7.5 and 8.0, respectively, indicating a slight increase in toxicity with increasing pH. The NOEC values were 4.0, 4.0, 2.0 and 2.0 mg Zr/L, respectively. These NOEC values were deduced from the optical density measurements (at 440 nm) reported in the publication, i.e., the NOEC values were biomass-based too. Biomass-based effect concentrations are usually somewhat lower than growth rate-based effect concentrations. It should be kept in mind that growth rate-based effects are considered more ecologically relevant. Finally, in this study, it was also demonstrated that nitrate levels equal to those added during the zirconium oxynitrate assays did not affect algal growth rate. The observed effects can hence be ascribed to the molecule zirconium oxynitrate as such, to the element Zr, or to secondary effects (based on the well-known behavior of complexation with phosphates, see the other endpoint study records).

Reference 7

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.

Description of key information

A weight of evidence approach was performed using five studies: two studies with the read across substance 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), one with a reaction mass of cerium dioxide and zirconium dioxide, which is an insoluble zirconium compound (Peither, 2009), and one with zirconium dinitrate oxide itself (Martins et al., 2007). 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.

Additional information

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

The study of Martins et al. (2007) is the only study on zirconium dinitrate oxide. In this study, algal growth inhibition experiments were performed with Pseudokirchneriella subcapitata and Pandorina morum at pH 6.5, 7.2, 7.5 and 8.0. For P. subcapitata, 96-h EC50 values (biomass-based) were obtained of 9.8, 7.9, 7.3, and 6.2 mg Zr/L, respectively (corresponding to 24.84, 20.02, 18.50 and 15.71 mg zirconium dinitrate oxide/L), whereas the NOEC values were determined to be 4.0, 4.0, 2.0, and 2.0 mg Zr/L (corresponding to 10.14, 10.14, 5.07, 5.07 mg zirconium dinitrate oxide/L), respectively, at these pH levels. For P. morum, 96-h EC50 values were 4.6, 8.1, 3.2, and 2.9 mg Zr/L (corresponding to 11.66, 20.53, 8.11, 7.35 mg zirconium dinitrate oxide/L), respectively, whereas the NOEC values were determined to be respectively 0.5, 8.0, 0.5, and 0.5 mg Zr/L at these pH levels (corresponding to 1.27, 20.28, 1.27 and 1.27 mg zirconium dinitrate oxide/L). No analytics were performed during this study to validate test concentrations, neither were phosphate measurements performed throughout the study. Therefore, no conclusions can be drawn on the extent to which the observed adverse effects were due to direct toxicity of the test substance or rather to potential secondary effects such as phosphate deprivation due to precipitation of phosphates with zirconium, the latter being observed in other studies performed with zirconium compounds. Therefore, the study was considered reliable with restrictions (Klimisch 2) and to be used only in a weight of evidence approach together with other information.

Four read across studies were included in the weight of evidence approach in order to be able to draw conclusions on the toxicity of zirconium dinitrate oxide to aquatic plants. The most recent test was performed using the read across substance zirconium dichloride oxide (Vryenhoef, 2014), another 'water soluble' zirconium compound with similar behaviour as zirconium dinitrate oxide. A range finding experiment indicated that no measurable zirconium could stay in solution (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).

The second study performed with the read across substance 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 (nominal concentration, no analytics performed).

Because at environmentally relevant conditions, zirconium precipitates out of solution (predominantly as zirconium hydroxide, zirconium dioxide, zirconium phosphate, and/or zirconium carbonate), read across from insoluble and/or sparingly soluble zirconium substances is considered acceptable too. Two additional read across studies with such compounds were therefore added to the weight of evidence approach to provide further evidence on the phosphate deprivation effect typically observed for zirconium substances. The first of these studies (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 of these studies (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.

Based on the above, it can be safely concluded that zirconium dinitrate oxide is not expected to cause any direct zirconium-related toxicity to aquatic plants, and that the observed effects in the study of Martins et al. (2007) are most likely due to phosphate deprivation, as was observed in studies with other zirconium compounds.