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Short-term toxicity to fish

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short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31 Jan 2013 - 07 April 2013
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to
OECD Guideline 203 (Fish, Acute Toxicity Test)
according to
EU Method C.1 (Acute Toxicity for Fish)
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
Details on sampling:
- Concentrations: concentrations of dissolved cerium 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 of 48 hours
- Sample storage conditions before analysis: stored at room temperature and 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 cerium ammonium nitrate corrected for purity) was prepared at the start of the test and before the test medium renewal by dispersing 3.02 g of the test item in 30 L of test water. This preparation was supported by ultrasonic treatment for 15 minutes. The pH of the dispersion was adjusted to 6.0 using 1 M sodium hydroxide solution, followed by intense stirring on a magnetic stirrer over 96 hours in the dark, to dissolve a maximum amount of the test item in the dispersion.

The long stirring period of 96 hours was selected according to the results of a pre-experiment conducted in Harlan Laboratories Study D60755 (in ISO medium, same ion composition in a 2-fold higher concentration compared to EPA medium), which showed that the maximum concentration of test item was reached after the stirring period of 96 hours.
After the 96-hour stirring period, the dispersion of the test item was filtered through a membrane filter (Schleicher & Schuell, Type NC45, pore size 0.45 µm

- Chemical name of vehicle (organic solvent, emulsifier or dispersant): No auxiliary solvent or emulsifier was used.
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): At the start of the first 48-hour test interval, the analytically determined concentrations of dissolved cerium in the test media (dilutions 1:16, 1:8, 1:4, 1:2 and the undiluted filtrate) were 0.0021, 0.0019, 0.0038, 0.0028 and 0.0098 mg/L, respectively, corresponding to anhydrous cerium ammonium nitrate concentrations of 0.0081, 0.0072, 0.015, 0.011 and 0.039 mg/L.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): no
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
- Common name: Oncorhynchus mykiss, rainbow trout
- Source: P. Hohler-Gasser, trout breeding station Zeiningen, 4314 Zeiningen / Switzerland
- Age at study initiation (mean and range, SD): not specified
- Length at study initiation (length definition, mean, range and SD): 4.9 ± 0.3 cm
- Weight at study initiation (mean and range, SD): mean body wet weight was 0.9 ± 0.2 g
- Feeding during test: no

- Acclimation period: one week
- Acclimation conditions (same as test or not): same as test
- Type and amount of food: commercial fish diet (HOKOVIT 502, 1.2 mm, supplied by H.U. Hofmann AG, 4922 Bützberg / Switzerland)
- Feeding frequency: During holding and acclimatization until one day before the start of the test
- Health during acclimation (any mortality observed): no
Test type:
Water media type:
Limit test:
Total exposure duration:
Remarks on exposure duration:
125 mg CaCO3/L
Test temperature:
13-14 °C
New test media:
0 h = 6.5 - 7.2 (before adjustment)
0 h = 6.0 (after adjustment)
48 h = 6.2 - 7.3 (before adjustment)
48 h = 6.0 (after adjustment)

Old test media:
24 h = 6.1-6.3
48 h = 6.2-6.5
72 h = 5.5 - 6.4
96 h = 6.2-6.4
Dissolved oxygen:
9.4 mg/L (corresponding to 89% saturation)
during test: between 9.5-9.8 mg/L
not applicable
Nominal and measured concentrations:
At the start (0 hours):
Analytically determined concentrations in the control, dilutions 1:16, 1:8, 1:4, 1:2 and the undiluted filtrate were :
< LOQ, 0.00206, 0.00185, 0.00376, 0.00281 and 0.00984 mg dissolved Ce/L; corresponding to
Day 2, 48 hours:
Analytically determined concentrations in the control, dilutions 1:16, 1:8, 1:4, 1:2 and the undiluted filtrate were :
< LOQ, 0.00175, 0.00114, 0.00134, 0.00237, 0.0105 mg dissolved Ce/L; corresponding to
Day 2, 0h:
Analytically determined concentrations in the control, dilutions 1:16, 1:8, 1:4, 1:2 and the undiluted filtrate were :

At the end (96 hours):
Analytically determined concentrations in the control, dilutions 1:16, 1:8, 1:4, 1:2 and the undiluted filtrate were :
< LOQ, 0.0158, 0.0435, 0.0360, 0.162, corresponding to:
The biological results were based on the mean measured concentrations calculated as the arithmetic mean of the two geometric means determined for dissolved cerium and cerium ammonium nitrate concentrations measured at the start and end of the test medium renewal periods.
Details on test conditions:
- Test vessel: One glass test vessel
- Type (delete if not applicable): covered with plastic plates
- Material, size, headspace, fill volume: 15 liters of test medium was used for each treatment.
- Aeration: slight aeration
- Renewal rate of test solution (frequency/flow rate): every day
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- No. of vessels per vehicle control (replicates): 1
- Biomass loading rate: 1 g fish/L

- Source/preparation of dilution water: reconstituted water
- Alkalinity: 0.4 mmol/L
- Ca/mg ratio: 4:1
- Culture medium different from test medium: no

- Adjustment of pH: yes
- Photoperiod: A 16 hour light to 8-hour dark photoperiod, with a 30-minute transition period
- Light intensity: light intensity during the light period was approximately within the range of 140 to 480 Lux.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): mortality

- Spacing factor for test concentrations: 2
- Range finding study: yes
- Test concentrations: 1:1000, 1:100, 1:10
- Results used to determine the conditions for the definitive study: the selection of the test concentrations was based on the results of the range-finding test, no mortality seen in the treatments of the range finding test.
Key result
96 h
Dose descriptor:
Effect conc.:
0.14 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element (dissolved fraction)
Basis for effect:
Remarks on result:
other: 95% CL: 0.11-0.17
Key result
96 h
Dose descriptor:
Effect conc.:
0.53 mg/L
Nominal / measured:
Conc. based on:
test mat.
anhydrous Ce(NH4)3(NO3)6
Basis for effect:
Remarks on result:
other: 95% CL: 0.42-0.66
Details on results:
96 h NOEC and 96 h LC0 = 0.026 mg Ce/L (corresponding to 0.10 mg cerium ammonium nitrate/L)
96 h LC100 = 0.22 mg Ce/L (corresponding to 0.86 mg cerium ammonium nitrate/L)
Reported statistics and error estimates:
The LC50 and the 95%-confidence interval at the observation dates were calculated by Probit Analysis [Davies, 1971; Finney, 1971]. The lowest concentration (treatment 1:16) was excluded from the statistical evaluation, as it did not contribute to the dose-response relationship (no mortality observed after 96 hours up to treatment 1:4).
Validity criteria fulfilled:
The 96-hour LC50 was determined to be 0.14 mg/L for dissolved cerium corresponding to 0.53 mg/L for anhydrous cerium ammonium nitrate. Cerium was considered to have acute toxic effects on juvenile rainbow trout (Oncorhynchus mykiss).

Description of key information

One study (Hefner, 2014a) is included in this dossier and regarded as a key study (Klimisch score of 1). This study reported a 96-h LC50 of 0.14 mg dissolved Ce/L (corresponding to 0.53 mg (NH4)2Ce(NO3)6/L). 

Key value for chemical safety assessment

LC50 for freshwater fish:
0.14 mg/L

Additional information

Cerium ammonium nitrate had acute toxic effects on juvenile rainbow trout (Oncorhynchus mykiss) in a semi-static study performed according to OECD guideline 203 (Hefner, 2014a). The 96-hour LC50 was determined to be 0.14 mg/L for dissolved cerium with 95% confidence limits of 0.11 and 0.17 mg/L (corresponding to 0.53 mg/L of cerium ammonium nitrate with 95% confidence limits of 0.42 and 0.66 mg/L). Based on the results it can be considered that cerium ammonium nitrate is very toxic to fish.

Because of the discrepancy of this result with that for aquatic invertebrates (no adverse effects observed up to and including the limit nominal test dose of 100 mg cerium ammonium nitrate/L), it was investigated whether or not there might have been a confounding effect of ammonia. Nominally, 6.58 mg/L ammonia have been added to the test medium at the 100 mg/L cerium ammonium nitrate loading rate. At an average pH of 6.2 and temperature of 13.5°C, this corresponds to about 0.002 to 0.003 mg unionized ammonia/L (i.e., the toxic form, calculations performed using several tools, e.g., Unionized Ammonia Calculator v1.2, after original by Dr. Landon Ross, Florida Department of Environmental Protection). Studies on acute unionized ammonia toxicity to rainbow trout report the following levels of toxicity:

- 96-h LC50 of 0.16-1.1 mg/L for 1-d-old fry up to 4-y-o adults - fry were most sensitive (Thurston RV, Russo RC, 1983. Acute toxicity of ammonia to rainbow trout. Transactions of the American Fisheries Society 112, 696-704).

- 96-h LC50 of 0.15 mg/L for rainbow trout (USGS).

Therefore, it is highly unlikely that ammonia would have contributed to the observed effects in the test performed by Hefner (2014a). The remarkable difference in observed effects between the test with fish and the test with aquatic invertebrates may therefore be ascribed to a difference in exposure (dissolved cerium concentrations in the undiluted filtrate were between 0.00259 and 0.00338 mg Ce/L in the test with aquatic invertebrates whereas in the test with fish the undiluted filtrate yielded dissolved cerium concentrations between 0.322 and 0.566 mg/L (during the second 48-h renewal period)) and/or sensitivity of the test organisms.