Registration Dossier

Administrative data

Endpoint:
fish embryo acute toxicity (FET)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Study period:
2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
- Exposure duration in accordance with the 96-hour standard duration recommended in OECD 236 (Fish Embryo Acute Toxicity (FET) Test). - Analytical monitoring allowing to ascertain that actual concentrations are in adequation with nominal concentrations.

Data source

Reference
Reference Type:
publication
Title:
Effects of rare earth elements La and Yb on the morphological and functional development of zebrafish embryos
Author:
Jun’an Cui, Zhiyong Zhang, Wei Bai, Ligang Zhang, Xiao He, Yuhui Ma, Yan Liu, Zhifang Chai
Year:
2012
Bibliographic source:
Journal of Environmental Sciences 24(2): 209–213

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
This study investigated the effects of the rare earth element Yb on the morphological and functional development of zebrafish embryos by exposing them to a control and a range of Yb3+ concentrations (0.01 to 1.0 mmol/L) for 96 hours. Early life stage parameters such as egg and embryo mortality, gastrula development, tail detachment, eyes, somite formation, circulatory system, pigmentation, malformations, hatching rate, length of larvae and mortality were investigated.
GLP compliance:
no

Test material

Test material form:
solid
Details on test material:
- Name of test material: Ytterbium chloride hydrate (YbCl3·xH2O, x ≈ 6)
- Source of test material: Purchased from Afa Aesar, UK
- Purity: 99.9% when expressed as equivalent Rare Earth Oxide (REO)
No other information are available.

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
No sampling details indicated

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The stock solution of Yb3+ (10 mmol/L) was prepared by directly adding YbCl3·6H2O into Milli-Q water solution. A series of exposure liquid (0, 0.01, 0.1, 0.3, 0.5 and 1.0 mmol/L) were prepared by dilution with E3 medium. E3 medium (5 mmol/L NaCl, 0.17 mmol/L KC1, 0.33 mmol/L CaCl2 and 0.33 mmol/L MgSO4, pH (7.0 ± 1.0)) is the standard hatchery water for zebrafish eggs.
- Controls: Yes, test medium without test item.

Test organisms

Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
Zebrafish (D. rerio) were maintained in a closed flow through culture system filled with conditioned water (75 g NaHCO3, 18 g sea salt, 8.4 g CaSO4, per 1000 L; pH (7.0± 1.0); total hardness: 65 mg/L (as CaCO3); temperature: (27 ± 1)°C; conductivity: 485 μS/cm) in a 14 hr:10 hr light:dark cycle. They were fed twice daily with live brine shrimps (Artemia salina). On the evening before spawning, male and female fish (the number ratio of 2:1) were placed in a hatching box. Spawning was triggered once the light was turned on in the next morning and was completed within 1 hr. Viable eggs were collected and rinsed for at least three times with E3 medium (5 mmol/L NaCl, 0.17 mmol/L KC1, 0.33 mmol/L CaCl2 and 0.33 mmol/L MgSO4, pH (7.0 ± 1.0)), which is the standard hatchery water for zebrafish eggs.
To ensure the developmental synchronization at the beginning of exposure, the embryos at about 2.5–3.0 hr post fertilization (hpf) were sorted under a stereo microscope. Healthy embryos at blastula stage were then subjected to Yb3+ exposure.

Study design

Test type:
static
Water media type:
freshwater
Remarks:
E3 medium (5 mmol/L NaCl, 0.17 mmol/L KC1, 0.33 mmol/L CaCl2 and 0.33 mmol/L MgSO4, pH (7.0 ± 1.0)), which is the standard hatchery water for zebrafish eggs.
Limit test:
no
Total exposure duration:
96 h
Remarks on exposure duration:
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 (Bai et al., 2010).
Post exposure observation period:
no

Test conditions

Hardness:
not indicated
Test temperature:
27 ± 1°C
pH:
7.0 ± 1.0
Dissolved oxygen:
not indicated
Salinity:
not indicated
Conductivity:
not indicated
Nominal and measured concentrations:
Nominal concentrations: 0, 0.01, 0.1, 0.3, 0.5 and 1.0 mmol/L
Actual concentrations: 0, 0.00995, 0.0992, 0.298, 0.496 and 0.993 mmol/L
Details on test conditions:
- 14 hr:10 hr light:dark photoperiod
- The assay is mainly based on the embryo test procedure developed by Schulte and Nagel (1994). Briefly, twenty-four eggs were transferred into a 24-well multi-plates with one embryo per well. Twenty wells were filled with 2 mL of Yb3+ solutions, and the remaining four wells were filled with 2 mL 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)) medium to act as controls. All of the multi-plates were covered with transparent plastic films and placed in an illumination incubator at (27 ±1)°C with a 14 hr:10 hr light:dark photoperiod.
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
LC50
Effect conc.:
0.268 mmol/L
Nominal / measured:
not specified
Conc. based on:
element
Remarks:
Yb3+
Basis for effect:
other: hatching rate
Remarks on result:
other: equivalent to 46.375 mg Yb+3/L
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 0.01 - < 0.1 mmol/L
Nominal / measured:
not specified
Conc. based on:
element
Remarks:
Yb+3
Basis for effect:
mortality (fish)
Remarks on result:
other: equivalent to 1.73 to 17.30 Yb+3 mg/L
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
0.01 mmol/L
Nominal / measured:
not specified
Conc. based on:
element
Remarks:
Yb+3
Basis for effect:
other: body length
Remarks on result:
other: equivalent to 1.730 Yb+3 mg/L
Details on results:
Analytical monitoring has revealed that actual concentrations were in good agreement with nominal ones. Therefore, expressing the results based on either nominal or measured concentrations has no influence on the conclusions.

While the LC50 for hatching rate was directly given in the publication, the LC50 for mortality and the NOEC for body length were deduced from the graphes. In the publication, all data are expressed in mmol/L Yb3+. For the purpose of this registration dossier, they were converted in mg/L Yb3+. As the registered substance is anhydrous ytterbium trinitrate, the data were further converted as follows:

Hatching rate:
72h-LC50 = 0.268 mmol/L Yb3+
72h-LC50 = 46.375 mg/L Yb3+
72h-LC50 = 96.225 mg/L anhydrous ytterbium trinitrate

Mortality:
96h-LC50 = 0.01 to 0.1 mmol/L Yb3+
96h-LC50 = 1.73 to 17.30 mg/L Yb3+
96h-LC50 = 3.59 to 35.91 mg/L anhydrous ytterbium trinitrate

Body length:
96h-LC50 = 0.01 mmol/L Yb3+
96h-LC50 = 1.73 mg/L Yb3+
96h-LC50 = 3.59 mg/L anhydrous ytterbium trinitrate
Reported statistics and error estimates:
Statistical analysis was performed using the statistical package SPSS 10.0 for Windows (SPSS Inc., USA). Data are presented as the (mean ± SD). The data were tested for homogeneity and normality. If these assumptions were met, one-way analysis of variance (ANOVA) was performed. Otherwise, the non-parametric Kruskal-Wallis test was performed. Significance level was set as p < 0.05.

Applicant's summary and conclusion

Validity criteria fulfilled:
not applicable
Conclusions:
After exposure of embryos of Zebrafish to concentrations of Ytterbium chloride hydrate of 0, 0.01, 0.1, 0.3, 0.5 and 1.0 mmol/L, the 72h - LC50 (hatching rate) was estimated to be 0.268 mmol of Yb+3 /L (equivalent to 46.375 mg of Yb+3/L), the 96h - LC50 (mortality) was estimated to be between 0.01 and 0.1 mmol of Yb+3 /L (equivalent to 1.73 to 17.30 mg of Yb+3/L) and the 96h - NOEC (body length) was estimated to be 0.01 mmol of Yb+3 /L (equivalent to 1.73 mg of Yb+3/L).
Executive summary:

In a publication by Cui et al (2012), the effect of ytterbium chloride hydrate on the morphological and functional development of zebrafish (Danio rerio) embryos were studied. The embryos were exposed to concentration of Yb3+ of 0, 0.01, 0.1, 0.3, 0.5 and 1.0 mmol/L. Early life stage parameters such as egg and embryo mortality, gastrula development, tail detachment, eyes, somite formation, circulatory system, pigmentation, malformations, hatching rate, length of larvae and mortality were investigated. The 72h - LC50 (hatching rate) was reported to be 0.268 mmol of Yb+3 /L (equivalent to 46.375 mg of Yb+3/L), which corresponds to 96.225 mg ytterbium trinitrate/L. The 96h - LC50 (mortality) was reported to be between 0.01 and 0.1 mmol of Yb+3 /L (equivalent to 1.73 to 17.30 mg of Yb+3/L), which corresponds to 3.59 to 35.91 mg ytterbium trinitrate/L. The 96h - NOEC (body length) was reported to be 0.01 mmol of Yb+3 /L (equivalent to 1.73 mg of Yb+3/L), which corresponds to 3.591 mg ytterbium trinitrate/L. The author concluded that Yb3+ delayed zebrafish embryo and larval development, decreased survival and hatching rates, and caused tail malformation in a concentration-dependent way.