Gadolinium zirconium oxide (Gd2Zr2O7)

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-09-16 to 2016-12-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

23 March 2006

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source: H.C. Starck GmbH, Batch No.of test material: 150273V2
- Expiration date of the lot/batch:July 2021
- Purity test date: 98%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, dry, dark and tightly closed
- Stability under test conditions:Stable
- Solubility and stability of the test substance in the solvent/vehicle: 4 mg/L (according to OECD 105: loading rate of 100 mg/L and stirring for 72 h at 30 °C)

OTHER SPECIFICS: White Powder

Analytical monitoring:

yes

Details on sampling:

- Concentrations: NC = 0 mg/L, A=100 mg/L
- Sampling method: At the start of the test (0 h), the test solutions NC and A were sampled before division to the test vessels (each group: 3 samples of 10 mL). After 24 h and 48 h exposure, only the two additional vessels for chemical analyses of NC
and A were sampled (each group: 2 samples of 10 mL). At the end of the test (72 h), one replicate of the test vessels and the two additional vessels from NC and A were sampled (each group: 3 samples of 10 mL).
- Sample storage conditions before analysis: The samples were filtered (0.45 μm syringe filter, Whatman), filled in plastic centrifuge tubes and stored (< 2 weeks) in the fridge (5 °C ± 3 °C). Of each treatment, one of the samples from 0 h, 24 h, 48 h and 72 h was sent to the analytical laboratory (ASG) and analysed. The remaining samples were stored as retain samples in the fridge until finalization of the study.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The stock solution was prepared by adding 55.5 mg test item (including a factor of 1.11 to take into account the dilution caused by addition of the algal inoculum) to 500 mL test medium and shaking for 72 h using an overhead shaker at 22.1-22.8 °C in the dark. Subsequently, the test item solution was allowed to settle for ca. 0.5 h before use in the test as single test item loading rate.
- Controls: The negative control (NC; test medium) was treated in the same way as the test item solution.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Raphidocelis subcapitata (now classified as Pseudokirchneriella subcapitata)
- Strain: No. 61.81 SAG
- Source (laboratory, culture collection): From the Culture Collection of Algae at the University of Goettingen and was cultivated in suspension culture. The strain used for this study has been cultured at Hydrotox GmbH since Mai 2016.
- Age of inoculum (at test initiation): 4 d
- Method of cultivation: Holm-Hansen medium under axenic conditions

ACCLIMATION
- Acclimation period: 4d
- Culturing media and conditions (same as test or not): culturing - Holm-Hansen medium, test - OECD TG 201 medium; other conditions are the same (23.2 - 23.3 °C and 73.2 μE m-² s-1 ± 5.6 % continuous lighting)
- Any deformed or abnormal cells observed: no

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

The temperature during the exposure was 23.1 - 23.3 °C

pH:

The pH was 7.8 - 7.5 in the control and 7.7 - 7.4 in the test item treatment.

Salinity:

not applicable

Nominal and measured concentrations:

nominal: NC = 0; A = 100 mg/L
measured: NC < LOQ (1µg/L); A < LOQ (1µg/L)

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer glass flasks 100 mL, Schott, Mainz
- Type (delete if not applicable): closed; The test vessels were sealed with cellulose stoppers.
- Fill volume: 50 mL
- Aeration: no
- Initial cells density: 7 × 10^3 cells/mL
- Control end cells density: 4.413 x 10^5 cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates):6

GROWTH MEDIUM
- Standard medium used: yes (OECD TG 201 medium)

TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: Holm-Hansen medium

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH:no
- Photoperiod:continous lighting
- Light intensity and quality: Mean light intensity was 73.2 μE m-² s-1 ± 5.6 %

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Measurement of chlorophyll fluorescence to be converted into cell counts per ml by means of a correlation factor. The correlation factor is based on cell counts with a Coulter Counter and the corresponding fluorescence is measured with a microplate fluorescence reader.
- Chlorophyll measurement: Chlorophyll fluorescence (excitation wavelength of 465 nm, emission wavelength of 670 nm)

TEST CONCENTRATIONS
- Justification for using less concentrations than requested by guideline: A preliminary test without GLP was performed before start of this GLP-study. Nominal loading rates of 45, 90 and 180 mg/L test item were tested and resulted in inhibition of -11.3 to -12.6 % for yield and -2.3 to -2.6 % for growth rate.
- Range finding study: yes, not GLP-conform
- Test concentrations (in range finding test): 45, 90 and 180 mg/L
- Results used to determine the conditions for the definitive study: Yes

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- Unusual cell shape: no (The morphology of the algal cells in the test loading rates as well as in the control showed no obvious abnormality.)
- Colour differences: not reported
- Flocculation: not reported
- Adherence to test vessels: not reported
- Aggregation of algal cells: not reported
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no

The inhibition of yield in the test item treatments, in relation to the control, is presented in table 2. Respectively, the inhibition of the specific growth rate in the test item treatments, in relation to the control, is presented in table 3. The algae cell concentrations in the control (NC) and the test item treatment are presented in the following illustration part of this documentation (figure 1). The effect loading rates after 72 h exposure are calculated with the statistical software ToxRat Professional 3.2.1 (see table 4).

Table 2: Inhibition of yield after 24 h, 48 h and 72 h exposure.

Nominal test item loading rate [mg/L]	Inhibition of yield [%]
	24 h	48 h	72 h
NC	--	--	--
100	-4.0	8.5	-1.1

Table 3: Inhibition of growth rate after 24 h, 48 h and 72 h exposure.

Nominal test item loading rate [mg/L]	Inhibition of growth rate [%]
	24 h	48 h	72 h
NC	--	--	--
100	-7.6	0.4	-1.6

Table 4: Effect loading rates for yield and growth rate at the end of the Test (72 h).

Lowest/ No Observed Effect Loading Rate	Nominal test item loading rate [mg/L]
	Yield	Growth rate
LOELR	> 100	> 100
NOELR	≥ 100	≥100

Validity criteria fulfilled:

yes

Remarks:

Biomass in the control increased by factor 97.8 (≥ 16). The mean coefficient of variation for section-by-section growth rate in control is 30.1 % (≤ 35 %). The coefficient of variation of average specific growth rate in control replicates is 3.8 % (≤ 7 %)

Conclusions:

The test item is no toxic ultures of Raphidocelis subcapitata.

Executive summary:

In a 72 h growth inhibition test, cultures of Raphidocelis subcapitata (Strain No. 61.81 SAG, now classified as Pseudokirchneriella subcapitata) were exposed to Gadolinium zirconium oxide at nominal loading rates of 0 (control) and 100 mg/L under static conditions in accordance with the OECD Guideline 201 (March 2006) and under GLP. The test design was chosen as limit test, as a pre-test (not GLP conform) with 45, 90 and 180 mg/L nominal concentrations showed no growth inhibition.

Within the limit test, the test item loading rate of 100 mg/L resulted in an inhibition of yield of -1 % (after 72 h) and an inhibition of the growth rate of -1.6 % (after 72 h) which is statistically not a significant difference to the control.

As no effect was observed the LOELR for both, growth rate and yield, must exceed 100 mg/L. The NOELR, for yield and growth rate, exceeds or equals 100 mg/L.

The measured Gd- and Zr-concentrations in the test item treatment were below the limit of quantification (< 1 μg/L).

Based on the results of this study, Gadolinium zirconium oxide would not be classified as toxic to R. subcapitata (now classified as P. subcapitata) in accordance with the classification system of CLP. This toxicity study is classified as acceptable and satisfies the guideline requirement for an aquatic toxicity study to algae.

Results synopsis:

Test organism: Raphidocelis subcapitata (now classified as Pseudokirchneriella subcapitata)

Test type: static

Growth rate:

72 h LOELR:  > 100 mg/L

72 h NOELR:  >= 100 mg/L

Yield:

72 h LOELR:  > 100 mg/L

72 h NOELR:  >= 100 mg/L

Endpoint(s) effected:  Growth rate and yield

Description of key information

A limit test in accordance with the OECD Test Guideline 201 (23 March 2006) did neither show a significant inhibition of yield (-1.1 %) nor inhibition of growth (-1.6 %) caused by Gadolinium zirconium oxide on the algae Raphidocelis subcapitata (Strain No. 61.81 SAG, now classified as Pseudokirchneriella subcapitata). The results based on the growth rate are as follows: NOELR ≥ 100 mg/L, LOELR > 100 mg/L. The results on yield are similar: NOELR ≥ 100 mg/L, LOELR > 100 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

100 mg/L