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Short-term toxicity to aquatic invertebrates

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short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 Sept 2012 - 15 Mar 2013
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
Details on sampling:
- Concentrations: Concentrations of dissolved Ce were determined in at least one of the triplicate samples from each treatment per sampling time.
- Sampling method: Triplicate samples were taken from each treatment at the start and end of each test medium renewal period (after filtration through a membrane filter, Whatman, Type NC45, pore size 0.45 µm).
- Sample storage conditions before analysis: All samples are stored at room temperature in the dark after sampling until analysis.
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Due to the low solubility of the test item in the test media, a dispersion with the loading rate of 100 mg/L (based on anhydrous cerium trinitrate) was prepared at the start of the test and before each test medium renewal by dispersing 133.3 mg of the test item (dosing range: 133.2-133.3 mg - to take into account the water content in the test item) in 1000 mL of test water. This preparation was supported by ultrasonic treatment for 15 minutes and intense stirring on a magnetic stirrer over 3 hours in the dark, to dissolve a maximum amount of the test item in the dispersion. No auxiliary solvent or emulsifier was used.

An optimal stirring period of 3 hours was chosen based on the results of a pre-experiment, which showed that the solution equilibrium was reached after this time. In this pre-experiment, similar concentrations of dissolved elemental cerium were analytically measured in filtrates after stirring for 3, 24 and 96 hours.

After stirring and pH adjustment, the dispersion of the test item was filtered through a membrane filter (Whatman, Type NC45, pore size 0.45 µm). The undiluted filtrate was used as highest concentrated test medium and as a stock solution for preparation of the test media with lower test concentrations. For preparation of the latter, the filtrate was diluted with pH adjusted test medium.
Test organisms (species):
Daphnia magna
Details on test organisms:
- Common name: Daphnia magna
- Strain: Straus
- Source: University of Sheffield / UK in 1992
- Age at study initiation (mean and range, SD): 6-24 hours (not a first brood progeny)
- Feeding during test: no
- Method of breeding: clone has been bred at Harlan Laboratories in reconstituted water of the quality identical to the water quality used in the test

- Type and amount of food: green algae Desmodesmus subspicatus CHODAT, Strain No. 86.81 SAG, supplied by the Collection of Algal Cultures or a mixture of this algal suspension and a commercial fish diet
- Feeding frequency: daphnids are generally fed three times a week
- Health during acclimation (any mortality observed): good. No mortalities observed
Test type:
Water media type:
Limit test:
Total exposure duration:
48 h
250 mg CaCO3/L
Test temperature:
New test media:
0 h = 6.0-6.4
24 h = 5.9-6.2
Old test media:
24 h = 6.8-7.0
48 h = 6.1-7.1
Dissolved oxygen:
at least 8.1 mg/L
not applicable
Nominal and measured concentrations:
Analytically determined concentrations of dissolved cerium in the test media (control, dilutions 1:16, 1:8, 1:4, 1:2, and the undiluted filtrate) were as follows:
Day 0, 0 h (start of experiment) = < LOQ, 0.294, 0.664, 1.37, 2.82, and 5.58 mg Ce/L
Day 1, 24 h (end of 1st renewal period) = < LOQ, 0.203, 0.124, 0.130, 0.305, and 2.24 mg Ce/L
Day 0, 0 h (start of 2nd renewal period) = < LOQ, 1.95, 3.97, 8.02, 17.2, and 33.4 mg Ce/L
Day 2, 24 h (end of 2nd renewal peirod) = < LOQ, 0.0266, 0.109, 1.56, 9.80 and 30.3 mg Ce/L
Over the test medium renewal periods of 24 h, the concentrations of dissolved Ce decreased (recovery 1.0 to 91% of initially measured concentrations, on average 11 to 65%). Further, the dissolved Ce levels were overall higher during the second renewal period.
Biological results were based on mean measured concentrations calculated as the arithmetic mean of the two geometric means determined for dissolved Ce measured at the start and end of each test medium renewal period.
Mean measured concentrations were as follows (not mentioning the control, for which dissolved Ce was < LOQ):
0.2, 0.5, 2.0, 7.0 and 18.0 mg Ce/L
Details on test conditions:
- Test vessel: 100-mL glass beakers filled with 50 mL of test medium
- Type (delete if not applicable): open
- Aeration: The test water was aerated prior to the start of the study. No aeration during the study.
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

- Source/preparation of dilution water: Reconstituted test water according to ISO 6341
- Ca/mg ratio: 4:1
- Culture medium different from test medium: no

- Adjustment of pH: 1) adjustment of dispersion to pH 6.0 after the 3-h stirring period using 1 M NaOH, 2) adjustment of test medium (for dilution of the filtrate) to pH 6.0 using 1 M HCl
- Photoperiod: A 16-hour light to 8-hour dark cycle with a 30-minute transition period
- Light intensity: Light intensity during the light period was approximately between 390 and 560 Lux.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): immobilisation, daily

- Spacing factor for test concentrations: 2 (spacing factor for serial dilutions)
- Range finding study: yes
- Test concentrations range finding study: undiluted filtrate, 1:10, 1:100, 1:1000 dilution, and control
- Results used to determine the conditions for the definitive study: 0% immobilisation at 1:10 dilution, 100% immobilisation in undiluted filtrate
Reference substance (positive control):
potassium dichromate
48 h
Dose descriptor:
Effect conc.:
6.9 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element (dissolved fraction)
Basis for effect:
Remarks on result:
other: 95% CL = 5.9-8.2
48 h
Dose descriptor:
Effect conc.:
16 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
other: cerium trinitrate, anhydrous
Basis for effect:
Remarks on result:
other: 95% CL = 14-19
Details on results:
48-hour EC0 and NOEC of dissolved cerium were both 2.0 mg/L, since no toxic effect was observed up to and including this test concentration. 48-hour EC100 of dissolved cerium was 18 mg/L.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- 48-hour EC50: 0.94 mg/L (within the internal historical range: 48-hour EC50 from 2000 to 2012: 0.43 - 1.1 mg/L)
Reported statistics and error estimates:
NOEC, EC0 and EC100 were determined directly from the raw data.
24-h EC50 could not be calculated due to absence of toxicity and was therefore also determined directly from the raw data.
48-h EC50 and 95% confidence limits were calculated using Weibull Analysis with linear weighted regression.
The two lowest treatments were excluded from the statistical evaluation, as they did not contribute to the dose-response relationship.
Validity criteria fulfilled:
The test item had acute toxic effects on Daphnia magna. Based on mean measured concentrations, the 48-hour EC50 was determined to be 6.9 mg/L for dissolved cerium with 95% confidence limits of 5.9 and 8.2 mg/L and 16 mg/L for anhydrous cerium trinitrate with 95% confidence limits of 14 and 19 mg/L.
Executive summary:

The acute toxicity of cerium trinitrate to Daphnia magna was determined in a 48-hour semi-static test according to the OECD guideline 202 and in compliance with the principles of Good Laboratory Practice. Juvenile daphnids were exposed to a control treatment and the test chemical at various concentrations (a dispersion with the loading rate of 133 mg/L (corresponding to 100 mg/L of anhydrous cerium trinitrate) was used as stock solution for test media preparation; the undiluted filtrate and dilutions 1:2, 1:4, 1:8 and 1:16 of this undiluted filtrate were used as test media). Organisms were observed after 24 and 48 hours for immobility. After 48 hours of exposure, no immobilized test organisms were recorded in the control and up to and including the mean measured concentration of 2.0 mg/L of dissolved cerium, corresponding to 4.6 mg/L of anhydrous cerium trinitrate. At the mean measured concentration of 7.0 and 18 mg/L of dissolved cerium (corresponding to 16 and 41 mg/L of anhydrous cerium trinitrate), 50% and 100% of the daphnids were found to be immobile, respectively. The 48-h EC50 was determined to be 6.9 mg/L for dissolved cerium and 16 mg/L for anhydrous cerium trinitrate.

Description of key information

The key study of Hefner (2014), in which Daphnia magna was exposed for 48 h to Ce(NO3)3, yielded a 48-h EC50 of 6.9 mg Ce/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Dose descriptor:
Effect concentration:
6.9 mg/L

Additional information

The key study (Hefner, 2014) was a 48-h study with Daphnia magna in which Ce(NO3)3 was used as test item and dissolved cerium concentrations were analytically monitored during the test. The 48-h EC50 was calculated based on mean measured concentrations and was 6.9 mg Ce/L. These results are considered as the key values for this endpoint.