Europium trinitrate

Short-term toxicity to fish

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:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Remarks:

A Klimisch score of 3 was attributed for the following reasons: - Too short exposure duration (i.e. 72 hours) compared to the 96-hour standard duration recommended in OECD 236 (Fish Embryo Acute Toxicity (FET) Test). - No analytical monitoring while we know that rare earth nitrates do not dissolve easily in aquatic media used for ecotoxicological tests; which results in actual concentrations that are often well lower than nominal concentrations. - No indication about the statistical significance of the differences among treatments; thus not possible to derive NOEC for the different endpoints (hatching time, standard length at hatching, development and functionality of embryonic heart). However, it was decided to include this publication into IUCLID because it confirms that rare earth nitrates exert toxicity on fish as already evidenced with other rare earth nitrates (Ce, La, Y, Gd, Pr, Dy, Nd).

Data source

Reference

Reference Type:

publication

Title:

Toxic effects of Europium chloride on developing zebrafish (Danio rerio) embryos

Author:

LAZARIS D., LIASKO R., EONARDOS I., EVANGELOU A., KALFAKAKOU V.

Year:

2012

Bibliographic source:

Journal of Biological Research-Thessaloniki 18: 291-296

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

This study evaluates the toxicity of europium (tested as europium chloride hexahydrate, EuCl3.6H2O) to zebrafish embryos. Two exposure conditions were tested: (1) continuous exposure for 72 hours post-fertilisation, (2) exposure only for the first 10 hours after fertilisation.

GLP compliance:

not specified

Test material

Test material information

Test material form:

solid

Details on test material:

- Name of test material: Europium chloride hexahydrate
- Formula: EuCl3.6H2O
- CAS number: 13759-92-7
- Source: obtained from Aldrich Chem.
No other information are available.

Sampling and analysis

Analytical monitoring:

no

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION:
- Method: No data
- Controls: Yes
No further data

Test organisms

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebrafish.
- Source: Breeding was performed on fish obtained from wild stocks and maintained in the laboratory for at least three generations. Fertilised eggs were obtained from reproducing sexually mature zebrafish according to the procedures described by Westerfield (1995).
- Age at study initiation: Fertilised eggs collected within two hours after spawning.
- Method of breeding: Sexually mature zebrafish were kept in 40 L full glass aquaria with a flow-through system (flow rate 10-20 L.hr–1, 28.5±1ÆC, pH 7.2±0.2 and total hardness 24ÆdH). Light and dark periods were 14 and 10 hrs, respectively, and fish were fed with commercially available artificial diet (ZM-400, Tetra Werke, Melle, Germany) twice per day.
No further data.

FEEDING DURING TEST
- Food type: No (exposure of embryos).

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

Two exposure durations were tested: (1) continuous exposure for 72h post-fertilisation, (2) exposure only for the first 10h after fertilisation. In the second case, after 10h-exposure, the embryos were placed into clean fresh water.

Test conditions

Hardness:

No data

Test temperature:

28.5 ± 1 °C

pH:

No data

Dissolved oxygen:

No data

Salinity:

No data

Conductivity:

No data

Nominal and measured concentrations:

Nominal concentrations: 0, 0.05, 0.5, 5, 50, 100, 200, 320, 400 and 500 mg Eu/L. All concentrations were tested under the conditions of continuous 72h-exposure. Only the concentrations of 200, 320, 400 and 500 mg Eu/L were tested under the conditions of 10h-exposure post fertilisation.
Measured concentrations: not available (no analytical monitoring).

Details on test conditions:

TEST SYSTEM
- Test vessel: Petri dish.
- No. of organisms per vessel: 40 embryos.
- No. of vessels per concentration (replicates): 3 replicates.
- No. of vessels per control (replicates): 3 replicates.

TEST MEDIUM / WATER PARAMETERS
No data.

OTHER TEST CONDITIONS
No data.

EFFECT PARAMETERS MEASURED
- Mortality recorded every hour.
- Hatching recorded every hour.
- Morphometric analysis performed at the end of the test: A total of nine raw distances [eye diameter, mouth to posterior margin of pericardial cavity, posterior margin of pericardial cavity to urogenital opening, urogenital opening to posterior tip of notochord, the highest point of head (dorsally from rhombencephalon) to posterior tip of notochord, posterior tip of notochord to anterior margin of dorsal fin, posterior tip of notochord to posterior margin of dorsal fin, mouth to the highest point of head, posterior margin of pericardial cavity to the highest point of head] along with eight areas (eye, yolk sac, dorsal-caudal fin, ventral fin, notochord, otic capsule, otoliths and body area) were recorded.
- Presence of well-developed heart assessed at the eye-stage (48h post fertilisation).
- Heart rate (beats per minute) was measured at 48 and 72 hrs after fertilisation.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: No regular spacing factor.
No further data.

Reference substance (positive control):

not specified

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

LL50

Effect conc.:

277.1 mg/L

Nominal / measured:

nominal

Conc. based on:

other: europium

Basis for effect:

mortality (fish)

Remarks on result:

other: (95% CI: 233.9 - 323.0 mg/L) Result for a continuous 72h-exposure

Effect concentrations 2

Duration:

10 h

Dose descriptor:

LL50

Effect conc.:

448.6 mg/L

Nominal / measured:

nominal

Conc. based on:

other: europium

Basis for effect:

mortality (fish)

Remarks on result:

other: (95% CI: 366.4 - 574.9 mg/L) Result for an exposure the first 10h after fertlisation

Details on results:

MORTALITY RATES:

- Control: 0 % (continuous 72h-exposure) and 0 % (exposure the first 10h after fertilisation).
- 0.05 mg Eu/L: 0 % (continuous 72h-exposure) and not evaluated (exposure the first 10h after fertilisation).
- 0.5 mg Eu/L: 0 % (72h) and not evaluated (10h).
- 5 mg Eu/L: 0 % (72h) and not evaluated (10h).
- 50 mg Eu/L: 0 % (72h) and not evaluated (10h).
- 100 mg Eu/L: 5 % (72h) and not evaluated (10h).
- 200 mg Eu/L: 18.2 % (72h) and 8.6 % (10h).
- 320 mg Eu/L: 49.5 % (72h) and 24.1 % (10h).
- 400 mg Eu/L: 78.9 % (72h) and 38.3 % (10h).
- 500 mg Eu/L: 100 % (72h) and 54.4 % (10h).

When exposed for 10h only, the death of embryos was significantly attenuated.

N.B. In the absence of analytical monitoring, it has to be noticed that the effect concentrations (LL50) are expressed on a nominal basis. Knowing that soluble salts of rare earth (like europium chloride) do not dissolve so easily in aquatic media used for ecotoxicological tests, it can be anticipated that actual concentrations should be lower than nominal concentrations.

HATCHING:

The mean hatching time was 56h post-fertilisation in control treatment. Exposure to europium significantly increased the median hatching time of alive and successfully hatched embryos, as well as the overall duration of hatching, with 72 hpf exposure resulting in a more profound delay. Under conditions of 10h-exposure, the median hatching time increased approximately up to 84h post-fertilisation at the concentration of 500 mg/L. Under conditions of 72h-exposure, the median hatching time increased approximately up to 85 h post-fertilisation at the concentration of 320 mg/L, after which extensive mortality of embryos was observed.

MORPHOMETRIC ANALYSIS:

- Standard length: The standard length of newly hatched larvae was significantly affected at high europium concentrations. After continuous 72h-exposure, a slight but significant decrease of mean standard length in comparison to the control group was observed at 100 mg/L of europium and became more pronounced at 320 mg/L. Above this concentration, the differences in standard length were not estimated due to the extensive mortality of embryos. A dose-dependent decrease in standard length was also observed after 10 hpf exposure. Moreover, high concentrations of europium, whether for 10h- or 72h-exposure, tend to produce more similar negative effects on the growth of embryos.

- Malformations: Malformations of body shape, eye and yolk sac were not seen in dead or alive embryos, except for heart aplasia in most dead embryos. When corrected for body size, the morphometric measurements were not significantly different even at highest europium concentrations used. Therefore, it is concluded that exposure to europium has no teratogenic effect and, more generally, does not result in significant alterations of the body shape.

HEART-RELATED PARAMETERS:

- Proportion of embryos without heart beat at 48h post fertilisation:
* Control: 1 % (continuous 72h-exposure) and 1 % (exposure the first 10h after fertilisation).
* 0.05 mg Eu/L: 4 % (continuous 72h-exposure) and not evaluated (exposure the first 10h after fertilisation).
* 0.5 mg Eu/L: 8 % (72h) and not evaluated (10h).
* 5 mg Eu/L: 16 % (72h) and not evaluated (10h).
* 50 mg Eu/L: 27 % (72h) and not evaluated (10h).
* 100 mg Eu/L: 29 % (72h) and not evaluated (10h).
* 200 mg Eu/L: 30 % (72h) and 18 % (10h).
* 320 mg Eu/L: 32 % (72h) and 26 % (10h).
* 400 mg Eu/L: not evaluated (72h) and 37 % (10h).
* 500 mg Eu/L: not evaluated (72h) and 49 % (10h).
Exposure to europium delayed the differentiation of the heart muscle depending on the concentration. At concentration of 320 ppm, the proportions of normal embryos were not significantly different between 10 and 72 hpf exposure times. At higher concentrations, most embryos were dead after continuous exposure, whereas for 10 hpf exposure the proportion of animals with normally developed heart continued to decline. Death of embryos was accompanied by complete heart aplasia in most cases.

- Heart rate: Even in the successfully heart developing embryos, the heart rate was affected by the presence of europium. The beats per minute decreased when europium concentrations increased under both exposure conditions.

Reported statistics and error estimates:

- Mortality of embryos was analysed by probit analysis (Finney, 1971) assuming the europium concentration as the single predictor and the natural response rate to be zero.
- The effects of europium on hatching rate of zebrafish were analysed using the Kaplan-Meier procedure (Hosmer & Lemeshow, 1999). Only successfully hatched larvae were included in the analysis. The overall statistical significance of the presence of europium (MantelCox test) as well as pairwise differences among hatching curves (log-rank test) were estimated. The median hatching time along with its confidence interval was determined for each concentration of europium.
- Differences in standard length at hatching were assessed by ANCOVA (n=40 per treatment) with exposure (10 or 72h post fertilisation) as fixed factor and log-transformed europium concentration as covariate. The standardised values of morphometric variables were subjected to multivariate analysis of variance as well as to discriminant analysis in order to assess eventual differences in body form between the groups of larvae exposed to different europium concentrations.
- Confidence intervals and differences for the proportions of embryos with well-developed heart at 48h post fertilisation were estimated using binomial test (n=154 - 331, according to various treatments). Changes in heart rate after exposure to europium were analysed by ANCOVA (type IV sum of squares) with the exposure time (10 or 72 h post fertilisation) and assessment time (48 and 72 h post fertilisation) as fixed factors and log-transformed europium concentration as covariate (n=40 per treatment).
- All analyses were performed using SPSS 16.0 software (SPSS Inc., Chicago, Illinois, USA).

Applicant's summary and conclusion

Validity criteria fulfilled:

not specified

Conclusions:

Under the conditions of this study, europium (tested as europium chloride hexahydrate, EuCl3.6H2O) presented significant toxic effects on survival, hatching time, standard length, heart development and heart rate of developing zebrafish eggs.

Executive summary:

This study evaluates the toxicity of europium (tested as europium chloride hexahydrate, EuCl3.6H2O) to zebrafish embryos. Two exposure conditions were tested: (1) continuous exposure for 72 hours post-fertilisation, (2) exposure only for the first 10 hours after fertilisation. Embryos were exposed to the following concentrations: 0, 0.05, 0.5, 5, 50, 100, 200, 320, 400 and 500 mg Eu/L. The whole concentrations were tested under the conditions of continuous 72h-exposure. Only the concentrations of 200, 320, 400 and 500 mg Eu/L were tested under the conditions of 10h-exposure post fertilisation. Regardless the exposure conditions, europium presented significant toxic effects on survival, hatching time, standard length, heart development and heart rate of developing zebrafish eggs. While the kind of effects are important because they give an indication about the endpoints targeted by the substance, it has to be kept in mind that effect concentrations (e.g. LL50) cannot be considered with confidence due to the absence of analytical monitoring. Knowing that soluble salts of rare earth (like europium chloride) do not dissolve so easily in aquatic media used for ecotoxicological tests, it can be anticipated that actual concentrations should be lower than nominal concentrations.