Zirconium oxide, hafnium and ytterbium doped

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

fish short-term toxicity test on embryo and sac-fry stages

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

unclear

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Principle of test: zebrafish embryo toxicity test according to Schulte and Nagel (1994)
- Short description of test conditions: one embryo per well in 24-well multi-plates; exposure with 0, 0.01, 0.1, 0.3, 0.5, 1.0 mmol/L Yb3+ for 96 h at 27+-1°C, 14 h:10h light:dark photoperiod.
- Parameters analysed / observed: gastrula development, tail detachment, eye development, somite formation, circulatory system, pigmentation, hatching rate, malformations, length of larvae and mortality.

GLP compliance:

not specified

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Ytterbium
chloride hydrate (YbCl3·xH2O, x ≈ 6), Alfa Aesar, UK;
- Purity, including information on contaminants, isomers, etc.: YbCl3 x H20 = 99.9% (REO), with no information on contaminants

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: no information
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: no information
- Stability in the medium, i.e. sensitivity of the test material to hydrolysis and/or photolysis: no information
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: no information
- Reactivity of the test material with the incubation material used (e.g. plastic ware): no information

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): no
- Preliminary purification step (if any): no
- Final concentration of a dissolved solid, stock liquid or gel: 10 mmol/L

OTHER SPECIFICS
- dilutions of 0, 0.01, 0.1, 0.3, 0.5 and 1.0 mmol/L were prepared with E3 medium (E3 (5 mmol/L NaC1, 0.17 mmol/L KC1, 0.33 mmol/L CaC12 and 0.33 mmol/L MgSO4, pH = (7.0 ± 1.0))

Analytical monitoring:

yes

Details on sampling:

not specified

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: not specified
- Controls: only negative control: E3 (5 mmol/L NaC1, 0.17 mmol/L KC1, 0.33 mmol/L CaC12 and 0.33 mmol/L MgSO4, pH = (7.0 ± 1.0))
- no vehicle
- No uniform test concentration separation factor
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): not specified

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebrafish embryos
- Strain: Danio rerio
- Source: not specified

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Numbers of parental fish (i.e. of females used to provide required number of eggs): not specified
- Method of collection of fertilised eggs: not specified
- Subsequent handling of eggs, embryos and larvae: viable eggs were collected and rinsed for at least three times with E3 medium

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

for parental fish: 65 mg/L; for fish embryos during test: not indicated; direct exposure to substance dissolved in E3 medium

Test temperature:

27+-1 °C

pH:

E3 medium: 7.0+-1.0, not specified if pH was monitored during test

Dissolved oxygen:

not specified

Conductivity:

for parental fish: 485 μS/cm; for fish embryos during test: not specified

Nominal and measured concentrations:

- nominal concentrations Yb3+: 0, 0.01, 0.1, 0.3, 0.5, 1.0 mmol/L
- measured concentrations Yb3+: 0, 0.00995, 0.0992, 0.298, 0.496, 0.993 mmol/L

Details on test conditions:

TEST SYSTEM
- Emybro cups (if used, type/material, size, fill volume): not applicable
- Test vessel: 24-well mulit-plates
- closed with transparent plastic films
- Material, size, headspace, fill volume: 2mL
- Aeration: not specified
- Renewal rate of test solution (frequency/flow rate): not specified
- No. of fertilized eggs/embryos per vessel: 1
- No. of vessels per concentration (replicates): 2 (20 wells were filled with 5 conc of Yb3+)
- No. of vessels per control (replicates): 4
- Biomass loading rate: not applicable

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: distilled water
- Culture medium different from test medium: no
- Intervals of water quality measurement: not indicated

OTHER TEST CONDITIONS
- Adjustment of pH: not indicated
- Photoperiod: 14hr/10 hr light:dark
- Light intensity: not indicated

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Direct observations were performed in the wells under a stereo microscope (20 × 1.5) connected to a camera device at specific timepoints (8, 24, 32, 48–60, 72, and 96 hr) during the period of 48–60 hr, and records were made every 2 hr.


RANGE-FINDING STUDY
- Test concentrations: 0, 0.01, 0.1, 0.3, 0.5, 1.0 mmol/L; unclear on which basis test concentrations were chosen and if a range-finding study was performed beforehand.

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC50

Remarks:

Yb3+

Effect conc.:

0.268 mmol/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

number hatched

Remarks on result:

other: Yb3+: 0.268 mmol/L x 174 g/mol = 0.047 g/L

Details on results:

- Mortality/survival at embryo and larval stages: at 0.5 mmol/L Yb3+ all larvae (n=6) died; basis of lethal effect was not described
- Overall mortality/survival: 100% mortality at 0.5 mmol/L Yb3+
- Days to hatch and numbers hatched: hatching rate of embryos was reported for 48 h to 96 h for every 2 h; exact numbers not indicated.
- Data for length and weight of surviving fish: solutions containing >= 0.1 mmol/L Yb3+ significantly decreased the body length of larvae.
- Type of and number with morphological abnormalities: body edema, bended tail, yolk arc edema; number not indicated
- Type of and number with behavioural abnormalities: not reported
- Other biological observations: no abnormality in other developmental endpoints on embryos seen (tail detachment, eye develoment somite formation, heartbeat, circulatory system, pigmentation, otic capsule)
- Effect concentrations exceeding solubility of substance in test medium: no information about solubility limit of Yb3+ in medium
- Incidents in the course of the test which might have influenced the results: none reported

Results with reference substance (positive control):

no positive control was used

Validity criteria fulfilled:

not applicable

Remarks:

study was conducted on basis of Schulte C, Nagel R, 1994. Testing acute toxicity in embryo of zebrafish, Brachydanio rerio as alternative to the acute fish test: Preliminary results. ATLA, 22(1): 12–19.

Conclusions:

The embryo toxicity test revealed that Yb3+ retarded the zebrafish embryos hatching (0.01–1.0 mmol/L), reduced the body length of larvae, killed the larvae died and caused malformation.

Executive summary:

The 96 h acute toxicity of Yb3+ to early life stage of Danio rerio was studied under static conditions. Embryos (2.5 - 3.0 h post fertilization) of Danio rerio were exposed to water (control) and Yb3+ of 0, 0.01, 0.1, 0.3, 0.5, 1 mmol/L nominal concentrations. Concentrations of Yb were measured in exposure solutions, but not during the test. Measured concentrations were: Yb3+: 0, 0.00995, 0.0992, 0.298, 0.496, 0.993 mmol/L.

The test system was maintained at 27±1ºC and a pH of 7±1.  The 72h EC₅₀ based on hatching rate, were 0.268 mmol/L for Yb3 +.  100% mortality was reported for 0.5 mmol/L Yb3+. The basis or indicator for mortality was not described. The OECD 236 lists the following indicators for lethality (i) coagulation of fertilised eggs, (ii) lack of somite formation, (iii) lack of detachment of the tail-bud from the yolk sac, and (iv) lack of heartbeat. None of these indicators were mentioned by Cui et al. (2011). Sublethal effects included decrease in body lenght of the larvae when exposed to solutions containing >= 0.1 mmol/L Yb3+ and occurence of body edema, bended tail, yolk arc edema. Exact numbers of affected larvae were not indicated. The most sensitive endpoint was not indicated.

This toxicity study is classified as supplementary and does not satisfy the guideline requirement for early life toxicity study with fish due to significant deficiencies such as reason for mortality missing, number of exposed embryos low (2 per concentration), solubility of test material in medium not indicated, reason for chosen test concentration not explained etc.

 

Results Synopsis

Test Organism: Danio rerio embryos 2.5 - 3 h post fertilization

Test Type: Static

EC50 (Yb3+) = 0.268 mmol/L = 0.047 g/L

Reference 2

Endpoint:

adult fish: sub(lethal) effects

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Description of key information

According to the results of the Transformation/Dissolution protocol (pH 8; ca. 22 °C; 7d; 100 mg/L loading), Zr and Hf are not released from the test item as measured concentrations were below the detection limit (see section 4.8 of the present dossier). Yb could be analytically verified at 0.069 μg/L and is hence the relevant component for the assessment of potential ecotoxicological effects. As Yb is only released in the low μg/L range and available short-term ecotoxicological studies with YbHfZrO2 in Daphnia (Knauer 2016, section 6.1.3) and algae (Knauer 2016, section 6.1.5) did not reveal any adverse effects at nominal concentrations of 100 mg/L, toxic effects on other fish are as well not expected and are considered highly unlikely. Consequently, no aquatic hazards were identified in the CSA (no PNECs were derived). Following the provisions of column 2 of chapter 9.1 on aquatic toxicity in Annex IX of the REACH regulation, "long-term testing shall be proposed ... if the CSA ... indicates the need to investigate further the effects on aquatic organisms." As no aquatic hazards were identified, further aquatic long-term tests are not needed. 

Cui et al. (2011) was identified as supporting study for long-term toxicity to fish. Zebrafish embryos were exposed to 0, 0.01, 0.1, 0.3, 0.5, 1.0 mmol/L of Yb3+ and La3+ for 96 h. Based on the hatching rate recorded at 72 h, an EC50 of 0.268 mmol/L (=0.047 g/L) was calculated for Yb3+. Although the study contains deficiencies regarding study design (e.g no positive control) and reporting (e.g. basis of mortality unclear), the EC50 for Yb3+ is clearly above the soluble part of Yb3+ from the target substance, 0.069 µg/L in the TD test, see above. Therefore, Yb3+ soluble concentration is expected to pose no hazard to aquatic organisms.