Reaction products of ricinoleic acid with 2-aminoethanol and maleic acid and sodium hydrogensulfite

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

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

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Control, 2.5, 8.0, 25, 80 and 249 mg test item/L (corresponding to 1.0, 3.2, 10, 32 and 100 mg solid content/L).
- Sampling method: For the determination of the actual test item concentrations in this semi-static test, duplicate samples were taken from each treatment just before the start of the first and of the last renewal periods (Days 0 and 3). At the end of these renewal periods (Days 1 and 4), additional samples were taken for determination of the stability of the test item during the renewal periods of 24 hours. All samples were taken from the approximate center of each aquarium without mixing the test medium.

- Sample storage conditions before analysis: All samples were taken from the approximate center of each aquarium without mixing the test medium, and were stored deep-frozen (at about -20 °C) immediately after sampling. Based on pre-experiments for investigation of the storage stability, the test item is sufficiently stable in the test water under the storage conditions.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test medium of the highest nominal concentration of 249 mg test item/L was freshly prepared by completely dissolving 1.494 g of test item (ranged from 1.4939 to 1.4944 g) in 6 liters of test water using ultrasonic treatment for 15 minutes and stirring for 15 minutes at room temperature. This highest test concentration was used in a series of dilution with test water to prepare the test media with the test concentrations as stated above. The test media were freshly prepared just before introduction of the fish (= start of the exposure) and before each test medium renewal.

- Controls: dilution water only
- Evidence of undissolved material (e.g. precipitate, surface film, etc): No remarkable observations were made concerning the appearance of the test media with the nominal test concentrations of 2.5 to 80 mg test item/L. All these test media were clear solutions throughout the test period. The media containing the nominal test item concentration of 249 mg test item/L were foamy (fresh media) or turbid and foamy solutions (aged media) throughout the test period. Measured concentrations within 80 - 120 % of nominal concentrations. Therefore, the turbidity is considered to have no effect on the test item concentration.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: zebra fish
- Strain: not provided
- Source: The fish were obtained from a breeding culture at Harlan Laboratories.
- Age at study initiation (mean and range, SD): not provided
- Length at study initiation (length definition, mean, range and SD): From the acclimated test fish batch, 10 fish were measured at the start of the test. The mean total body length (measured from the mouth to the end of the tail fin) of the fish was 3.0 ± 0.10 cm (Mean ± SD).
- Weight at study initiation (mean and range, SD): the mean body wet weight was 0.20 ± 0.02 g (Mean ± SD).
- Method of breeding: similar to test conditions
- Feeding during test: no

ACCLIMATION
- Acclimation period: Prior to test start, they were acclimated for one week to the test water and temperature.
- Acclimation conditions (same as test or not): similar
- Type and amount of food: During holding and acclimatization until one day before the start of the test, the fish were fed with a commercial fish diet (Tetra Min® Hauptfutter, supplied by TETRA-Werke, 49304 Melle / Germany).
- Feeding frequency: not reported
- Health during acclimation (any mortality observed):During holding and acclimatization, 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:

15 mg/L as CaCO3

Test temperature:

21-22 °C

pH:

7.2 to 7.5

Dissolved oxygen:

The oxygen concentration was at least 7.1 mg/L, corresponding to at least 79% oxygen saturation.

Nominal and measured concentrations:

The following nominal concentrations of Reaction products of ricinoleic acid with 2-aminoethanol and maleic acid and sodium hydrogensulfite were tested: 2.5, 8.0, 25, 80 and 249 mg test item/L (corresponding to 1.0, 3.2, 10, 32 and 100 mg solid content/L). Additionally, a control (test water without test item) was tested in parallel. In the analyzed test media samples (80 and 249 mg/L) from the start and end of the first and last test medium renewal periods the measured test item concentrations ranged between 83 and 103% of the nominal values. Consequently, the test water renewal ensured constant concentrations of the test item during the test and the reported biological results are related to the nominal test item concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel: glass vessels filled with 4 L test solution each
- Aeration: not reported, but DO concentration was within the validity range
- Renewal rate of test solution (frequency/flow rate): 24 h renewal interval.
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: 0.35 g/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted test water (AAP Medium) prepared according to the test guideline was used for this study.
- Culture medium different from test medium: no
- Intervals of water quality measurement: at every renewal of the freshly prepared and aged solutions

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: A 16 hour light to 8-hour dark photoperiod, with a 30-minute transition period was used
- 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) :
EC50 96 hours

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Justification for using less concentrations than requested by guideline: NA
- Range finding study
- Test concentrations: not reported
- Results used to determine the conditions for the definitive study: none

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

57 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95% C.I.:32 - 100 mg solid content/L

Details on results:

- Behavioural abnormalities:
- Observations on body length and weight: NA
- Other biological observations: In the control and at the test concentrations up to and including 80 mg test item/L (32 mg solid content/L), all fish survived until the end of the test and no visible abnormalities were observed in the test fish. At the highest test concentration of 249 mg test item/L (100 mg solid content/L), all test fish showed one visible abnormality (apathy). After 96 hours test duration, all seven test fish died at this test concentration.
- Mortality of control: none
- Other adverse effects control: none
- Abnormal responses: none
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Analsis of the test solutions confirmed correct sosing of the test solutions. No remarkable observations were made concerning the appearance of the test media with the nominal test concentrations of 2.5 to 80 mg test item/L. All these test media were clear solutions throughout the test period. The media containing the nominal test item concentration of 249 mg test item/L were foamy (fresh media) or turbid and foamy solutions (aged media) throughout the test period.
- Effect concentrations exceeding solubility of substance in test medium: no

Reported statistics and error estimates:

The 96-hour LC50 could not be calculated by Probit Analysis or Moving Average Interpolation due to a steep concentration-effect relationship. Instead, the 96-hour LC50 value was determined as a geometric mean value of the two consecutive test concentrations with 0 and 100% mortality, and the 95% confidence limits for the LC50 as the test concentrations with 0 and 100% mortality. The NOEC, LOEC, LC0 and LC100 were determined directly from the raw data.

Sublethal observations / clinical signs:

In the control and at the test concentrations up to and including 80 mg test item/L (32 mg solid content/L), all fish survived until the end of the test and no visible abnormalities were observed in the test fish. At the highest test concentration of 249 mg test item/L (100 mg solid content/L), all test fish showed one visible abnormality (apathy). After 96 hours test duration, all seven test fish died at this test concentration.

Validity criteria fulfilled:

yes

Conclusions:

The 96‑hour LC50of Reaction products of ricinoleic acid with 2-aminoethanol and maleic acid and sodium hydrogensulfite was calculated to be 57 mg solid content/L with a 95% confidence interval of 32 - 100 mg solid content/L

Executive summary:

In the Klimisch 1 GLP study from Kimmel (2013) the acute toxicity of Reaction products of ricinoleic acid with 2-aminoethanol and maleic acid and sodium hydrogensulfite zebra fish (Danio rerio) was determined in a 96‑hour semi-static test with daily test medium renewal according to the EU Commission Directive 92/69/, Part C.1, the Commission Regulation (EC) No 440/2008, Part C.1 and the OECD Guideline for Testing of Chemicals No. 203 (1992). The following nominal concentrations of Reaction products of ricinoleic acid with 2-aminoethanol and maleic acid and sodium hydrogensulfite were tested: 2.5, 8.0, 25, 80 and 249 mg test item/L (corresponding to 1.0, 3.2, 10, 32 and 100 mg solid content/L). Additionally, a control (test water without test item) was tested in parallel. In the analyzed biologically relevant test media samples (32 and 100 mg solid content/L) from the start and end of the first and last test medium renewal periods the measured test item concentrations ranged between 83 and 103% of the nominal values. Consequently, the test water renewal ensured constant concentrations of the test item during the test and the reported biological results are related to the nominal test item concentrations. In the control and at the test concentrations up to and including 80 mg test item/L (32 mg solid content/L), all fish survived until the end of the test and no visible abnormalities were observed in the test fish. At the highest test concentration of 249 mg test item/L (100 mg solid content/L), all test fish showed one visible abnormality (apathy). After 96 hours test duration, all seven test fish died at this test concentration.

The 96‑hour LC₅₀of Reaction products of ricinoleic acid with 2-aminoethanol and maleic acid and sodium hydrogensulfite was calculated to be 57 mg solid content/L with a 95% confidence interval of 32 - 100 mg solid content/L.

The results of this study are considered relevant and reliable for the risk assessment.

Description of key information

LC50: 57 mg/L

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

57 mg/L

Additional information

In the Klimisch 1 GLP study from Kimmel (2013) the acute toxicity of Reaction products of ricinoleic acid with 2-aminoethanol and maleic acid and sodium hydrogensulfite zebra fish (Danio rerio) was determined in a 96‑hour semi-static test with daily test medium renewal according to the EU Commission Directive 92/69/, Part C.1, the Commission Regulation (EC) No 440/2008, Part C.1 and the OECD Guideline for Testing of Chemicals No. 203 (1992). The following nominal concentrations of Reaction products of ricinoleic acid with 2-aminoethanol and maleic acid and sodium hydrogensulfite were tested: 2.5, 8.0, 25, 80 and 249 mg test item/L (corresponding to 1.0, 3.2, 10, 32 and 100 mg solid content/L). Additionally, a control (test water without test item) was tested in parallel. In the analyzed biologically relevant test media samples (32 and 100 mg solid content/L) from the start and end of the first and last test medium renewal periods the measured test item concentrations ranged between 83 and 103% of the nominal values. Consequently, the test water renewal ensured constant concentrations of the test item during the test and the reported biological results are related to the nominal test item concentrations. In the control and at the test concentrations up to and including 80 mg test item/L (32 mg solid content/L), all fish survived until the end of the test and no visible abnormalities were observed in the test fish. At the highest test concentration of 249 mg test item/L (100 mg solid content/L), all test fish showed one visible abnormality (apathy). After 96 hours test duration, all seven test fish died at this test concentration.

The 96‑hour LC₅₀of Reaction products of ricinoleic acid with 2-aminoethanol and maleic acid and sodium hydrogensulfite was calculated to be 57 mg solid content/L with a 95% confidence interval of 32 - 100 mg solid content/L.

The results of this study are considered relevant and reliable for the risk assessment.