2-ethylhexanoic acid, cerium salt

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 28 MAY 2009 to 06 JUL 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: For the determination of the actual test item concentrations, the concentration of cerium in the test media was analytically measured. In this semi-static test, duplicate samples were taken from each treatment just before test start of two test medium renewal periods (Day 0 and Day 3) and at the end of these renewal periods (Day 1 and Day 4). However, from the four highest test concentrations (undiluted filtrate and dilutions 1:3.2, 1:10, 1:32), the last samples were taken only from the first renewal period since all fish were dead at these concentrations before the next sampling date.
- Sampling method: All samples were taken from the approximate center of the aquaria without mixing of the test media.
- Sample storage conditions before analysis: Immediately after sampling, the samples were acidified with 10% (v/v) nitric acid (HNO3, 65% Suprapur®, Merck) to stabilize the samples during the storage period. The samples were stored in PE flasks at ambient temperature and protected from light until analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
While the test item is soluble in distilled water, the pre-experiments and the range-finding tests revealed a low water solubility in the reconstituted medium used for this study. For this reason, a dispersion with the loading rate of 144 mg/L (corrected for the water content of the test item of 30.54% and corresponding to a cerium nitrate concentration of 100 mg/L) was prepared at the start of the test and before each test medium renewal. At Day 0, 1584 mg of the test item (dosing range: 1584.9–1585.3 mg/L) were dispersed in 11000 mL of test water. At Day 1 and Day 2 of the test 432 mg of the test item (dosing range: 432.1–433.0 mg) were dispersed in 3000 mL of test water. At Day 3, 144.3 mg of the test item were dispersed in 1000 mL of test water (at Days 1 to 3 smaller volumes of test item suspension were prepared since the higher test media were no longer prepared as all test fish were dead). This preparation was supported by ultrasonic treatment for 15 minutes and intense stirring on a magnetic stirrer over 3 hours, to dissolve a maximum amount of the test item in the dispersion. No auxiliary solvent or emulsifier was used. The stirring period of 3 hours was chosen according to the results of a pre-experiment (without GLP) which showed that the solution equilibrium was reached after this time. After the 3-hour stirring period, the dispersion of the test item was filtered through a membrane filter (Schleicher & Schuell, Type NC45, pore size 0.45 μm). The pH of the undiluted filtrate was adjusted from 5.8 to 7.3 with a 1 M sodium hydroxide solution. The undiluted filtrate was used as the highest concentrated test medium and as a stock solution for preparation of the test media with lower test concentrations. For this preparation, the filtrate was diluted with test water. The test media were freshly prepared just before introduction of the fish (= start of the exposure) and before each test medium renewal. The preparation of the test media was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000.
- Differential loading: yes
- Controls: blank (test water without test item)
- Evidence of undissolved material (e.g. precipitate, surface film, etc): No remarkable observations were made concerning the appearance of the test media with the three lowest test concentrations (dilutions 1:320; 1:100 and 1:32). All these test media appeared clear solutions throughout the test medium renewal periods. At the start of the test medium renewal periods, the test media containing the three highest concentrations were clear (dilution 1:10) or slightly turbid (dilution 1: 3.2 and undiluted filtrate). At the end of the test medium renewal periods, test item precipitation was observed in the test media of these three highest test concentrations.

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: Rainbow trout
- Source: P. Hohler-Gasser, trout breeding station Zeiningen (Switzerland)
- Age at study initiation (mean and range, SD): no data
- Length at study initiation (mean +/- SD): 5.4 +/- 0.20 cm
- Weight at study initiation (mean +/- SD): 1.4 +/- 0.21 g
- Feeding during test : no

ACCLIMATION
- Acclimation period: the fish were held in the laboratory for more than twelve days without any medication. Prior to the test start, they were acclimated for 1 week to the test water and temperature.
- Type and amount of food: commercial fish diet (Hokovit 502)
- Feeding frequency: 5 times per week or on demand until one day before the start of the test
- Health during acclimation (any mortality observed): During holding and acclimatation no fish died in the test fish batch and all fish were healthy

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

125 mg/L as CaCO3

Test temperature:

14°C

pH:

6.4-7.3

Dissolved oxygen:

9.2 mg/L or higher

Salinity:

not applicable

Nominal and measured concentrations:

nominal concentrations: undiluted filtrate (loading rate: 144 mg/L corresponding to 100 mg/L cerium nitrate when corrected for the water content of the test item of 30.54%), dilution 1:3.2 (loading rate 45 mg/L), dilution 1:10 (loading rate 14.4 mg/L), dilution 1:32 (loading rate 4.5 mg/L), dilution 1:100 (loading rate 1.44 mg/L), dilution 1:320 (loading rate 0.45 mg/L)
Measured concentrations (based on test item): Undiluted filtrate (61 mg/L), dilution 1:3.2 (3.9 mg/L), dilution 1:10 (6.6 mg/L), dilution 1:32 (1.3 mg/L), dilution 1:100 (0.86 mg/L), dilution 1:320 (0.27 mg/L)

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Material, size, headspace, fill volume: glass test vessels filled with 15 L of test medium
- Aeration: The test water was aerated prior to the preparation of the test media until oxygen saturation was reached. During the test period, the test medium and the control were slightly aerated.
- Type of flow-through (e.g. peristaltic or proportional diluter): none (semi-static test)
- 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
- Biomass loading rate: 0.63 g fish wet weight / L


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted test water prepared by dissolving analytical grade salts in purified water.
- Total organic carbon: no data
- Particulate matter: no data
- Metals: no data
- Pesticides: no data
- Chlorine: no data
- Alkalinity: 0.4 mmol/L
- Ca/mg ratio: 4:1
- Na/K ratio: 10:1
- Conductivity: no data
- Bacteriological parameters: no data
- Culture medium different from test medium: no data
- Intervals of water quality measurement: The water temperature, pH values and oxygen concentrations were measured for each treatment with surviving fish at the start of the test and once daily during the test in the freshly prepared and old test media. At the same dates the appearance of the test media was recorded.

OTHER TEST CONDITIONS
- Adjustment of pH: After stirring and filtration of the stock solution (= undiluted filtrate), the pH was adjusted from 5.8 to 7.3 with a 1 M sodium hydroxide solution.
- Photoperiod: 16-hour light and 8-hour darkness with a 30-minute transition period
- Light intensity: 140-480 lux


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The test fish were observed after 3, 24, 48, 72 and 96 hours test duration for mortality and visible abnormalities. Dead fish were removed at least once daily and discarded.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2. This enlarged spacing factor between the test concentrations was chosen, because the concentration-effect relationship was rather flat and thus, a large concentration range had to be tested according to the results of the range-finding test.
- Range finding study : yes
- Test concentrations and Results used to determine the conditions for the definitive study: The selection of the test concentrations was based on the results of two previous main tests, range-finding tests and of pre-experiments to determine the dosage and water solubility of the test item (non-GLP).

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

0.13 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element

Remarks:

Ce

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

0.44 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: test material including the water content of 30.54%.

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

0.3 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

other: Ce (NO3)3

Basis for effect:

mortality (fish)

Details on results:

BIOLOGICAL RESULTS
- Mortality: At the two lowest test concentrations (dilutions 1:320 and 1:100), 2 and 5 fish died during the test period. At the dilution 1:32 and all higher treatments, all fish were dead until the end of the test period of 96 hours.
- Behavioural abnormalities: No visible abnormality was observed in the fish exposed to the lowest dilution (1:320). At dilutions 1:100, 1:32 and 1:10, abnormalities were observed from 24 hours of exposure, with fish presenting apathy, tumbling during swimming and staying at the water surface. At the highest concentrations (dilution 1:3.2 and undiluted filtrate), apathy and tumbling during swimming were observed from 3 hours of exposure.
- Observations on body length and weight: none
- Other biological observations: none
- Mortality of control: none
- Other adverse effects in control: none
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: See the § below: "APPEARANCE OF THE TEST MEDIUM"
- Effect concentrations exceeding solubility of substance in test medium: no

APPEARANCE OF THE TEST MEDIUM
No remarkable observations were made concerning the appearance of the test media with the three lowest test concentrations (dilutions 1:320, 1:100 and 1:32). All these test media appeared clear solutions throughout the test medium renewal periods. At the start of the test medium renewal periods, the test media containing the three highest test concentrations were clear (dilution 1:10) or slightly turbid (dilution 1:3.2 and undiluted filtrate). At the end of the test medium renewal periods, test item precipitation was observed in the test media of these three highest test concentrations.

ANALYTICAL MONITORING
At the start of the test medium renewals, the analytically determined mean concentration of cerium in the undiluted filtrate was 31.5 mg/L. This corresponds to 73.3 mg/L Ce(NO3)3 or 106 mg/L test item (including a water content of 30.54%). The measured starting concentrations of cerium in the lower test concentrations reflected the spacing factor of 3.2 between the test concentrations, demonstrating the correct preparation of the different dilutions. During the test medium renewal periods, a decrease of the cerium concentrations was observed.

Results with reference substance (positive control):

not tested

Reported statistics and error estimates:

The LC50 and the 95%-confidence interval at the observation dates were calculated by Probit Analysis. For calculation of the 96-hour LC50, the three highest test concentrations were not taken into account since these test concentrations were above the LC100 determined in this test. The calculations were based on the mean measured test concentrations. The mean measured concentrations during the different test medium renewal periods were calculated as geometric mean between the measurements from the start and the end of the different renewal periods. From the values obtained from each renewal period, the mean measured concentration over the test period of 96 hours was calculated as arithmetic mean. The NOEC, LOEC, LC0 and LC100 were determined directly from the raw data.

Sublethal observations / clinical signs:

Mortality and visible abnormalities observed in the test fish :

Treatment / Dilution	Mean measured test item Concentration (mg/L)	Number of abnormal and dead fish / number of dead fish Type of visible abnormalities
		Observation time
		3 hours	24 hours	48 hours	72 hours	96 hours
Control	-----	0 / 0	0 / 0	0 / 0	0 / 0	0 / 0
Dilution 1 :320	0.27	0 / 0	0 / 0	2 / 2	2 / 2	2 / 2
Dilution 1 :100	0.86	0 / 0	7 / 2 AP	7 / 4 AP, TS	7 / 4 AP	7 / 5 AP
Dilution 1 :32	1.3	0 / 0	7 / 5 TS	7 / 7	- / -	- / -
Dilution 1 :10	6.6	0 / 0	7 / 3 OB, TS	7 / 6 OB, TS	7 / 7	- / -
Dilution 1 :3.2	3.9	7 / 0 AP	7 / 7	- / -	- / -	- / -
Undiluted filtrate *	61	7 / 0 AP, TS	7 / 7	- / -	- / -	- / -
LC 50 95% C.I.		> 61	1.7	0.45	0.49	0.44
		-	n.d.	0.04-0.94	0.18-0.81	0.14 -0.74

* based on a test item loading rate of 144 mg/L

-/- : all fish dead

95% C.I.: 95% confidence interval of the LC50

n.d.: could not be determined

AP: Apathy

TS: Tumbling during swimming

OB: Fish mainly at the water surface

Validity criteria fulfilled:

yes

Conclusions:

The 96-h LC50 based on elemental concentration was 0.13 mg Ce/L.

Executive summary:

The acute toxicity of cerium trinitrate to rainbow trout (Oncorhynchus mykiss) was determined in a 96-hour semi-static test with a daily test medium renewal according to the EU Commission Directive 92/69/EEC, Part C.1 (1992), and the OECD Guideline for Testing of Chemicals No. 203 (1992).

Rainbow trout were exposed to control and test chemical at a loading rate of 144 mg/L (corresponding to 100 mg/L cerium nitrate when corrected for the water content of the test item of 30.54%) and dilutions 1:3.2, 1:10, 1:32, 1:100 and 1:320. The test fish were observed after approximately 3, 24, 48, 72 and 96 hours test duration for mortality and visible abnormalities.

In the control, all fish survived until the end of the test and no visible abnormalities were observed in the test fish. At the two lowest test concentrations (dilution 1:320 and 1:100), two and five fish died during the test period. At the dilution 1:32 and all higher treatments all fish were dead until the end of the test period of 96 hours.

The 96-hour LC50 to rainbow trout was determined to be 0.44 mg/L (mean measured concentration) based on test item, 0.30 mg/L (mean measured concentration) based on Ce(NO3)3, and 0.13 mg/L based on elemental Ce in the test medium.

Description of key information

The key study of Peither (2011), in which cerium was added as Ce(NO3)3, yielded a 96-h LC50 of 0.13 mg Ce/L for juvenile rainbow trout.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

LC50

Effect concentration:

0.13 mg/L

Additional information

The key study of Peither (2011) exposed juvenile rainbow trout for 96 h to Ce(NO3)3. Based on measured dissolved cerium concentrations, a 96-h LC50 of 0.13 mg Ce/L was obtained.