Reaction mass of sodium hydrogen N,N-dibutyl-10-(sulphonatooxy)octadecanamidate and sodium sulphooctadecanoate

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

fish short-term toxicity test on embryo and sac-fry stages

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-12-04 to 2012-12-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 212 (Fish, Short-term Toxicity Test on Embryo and Sac-Fry Stages)

Version / remarks:

adopted September 21, 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 7, 3.2, 1.5, 0.7 and 0.3 mg test item/L and a control

- Sampling method: For the determination of the stability of the test item under the test conditions and of the maintenance of the test item concentrations during the test period, duplicate test media samples were collected from all test concentrations and the control at the end of treatment periods A and B (after 24 hours of exposure).

- Sample storage conditions before analysis: At room temperature (20 ± 5 °C), under dark and dry conditions

Vehicle:

no

Details on test solutions:

The stock solution of nominal 50 mg/L was prepared by dissolving 50.9, 51.5, 51.5, 51.0, 50.9, 51.1, 50.8 and 50.0 mg test item into 1018, 1030, 1030, 1020, 1018, 1022, 1016 and 1000 mL test water, respectively, by intense stirring for 10 minutes. Then, adequate volumes of this stock solution were mixed into test water to obtain the desired test concentrations.
The test media were prepared just before introduction of the eggs (= start of the test) and test medium renewal was done on day 1, 2, 3, 4, 5, 6 and 7.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: zebra fish
- Source: University of Heidelberg, Germany.

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Subsequent handling of eggs, embryos and larvae: Freshly fertilized eggs (before beginning of gastrulation); unfertilized eggs not undergoing cleavage, showing obvious irregularities during cleavage or injuries of the chorion are discarded. As soon as possible after fertilization, the fertilized eggs were transferred into the test chambers.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

8 d

Hardness:

250 mg CaCO3/L

Test temperature:

25 to 26 °C

pH:

7.7 to 8.1

Dissolved oxygen:

87 to 100 % of the air saturation value

Nominal and measured concentrations:

Nominal concentration: 7, 3.2, 1.5, 0.7 and 0.3 mg test item/L and a control.
Measured concentration: freshly prepared: 83-85% of the nominal; start of the test: < 80% of the nominal (for the lower concentration levels); aged media: 50-53% of the nominal

Details on test conditions:

TEST SYSTEM
- Test vessel: 24 – well plates with 2 mL filling capacity
- Renewal rate of test solution: test medium renewal on days 1, 2, 3, 4, 5, 6 and 7
- No. of fertilized eggs/embryos per vessel: 10
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

TEST MEDIUM / WATER PARAMETERS
- Source of dilution water: Reconstituted Water (ISO Medium)

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h light: 8 h dark; 30 min dawn/dusk period was provided
- Light intensity: 880 to 890 lux

EFFECT PARAMETERS MEASURED:
Mortality, Hatching, Signs of Intoxication and Length

RANGE-FINDING STUDY
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

not required

Remarks:

according to the guideline a positive (reference) control has not to be performed

Key result

Duration:

8 d

Dose descriptor:

LC50

Effect conc.:

> 7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Egg-Mortality

Key result

Duration:

8 d

Dose descriptor:

LC10

Effect conc.:

> 7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Egg-Mortality

Key result

Duration:

8 d

Dose descriptor:

NOEC

Effect conc.:

>= 7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Egg-Mortality

Key result

Duration:

8 d

Dose descriptor:

LOEC

Effect conc.:

> 7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Egg-Mortality

Key result

Duration:

8 d

Dose descriptor:

LC50

Effect conc.:

7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Fry-Mortality

Duration:

8 d

Dose descriptor:

LC10

Effect conc.:

2.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Fry-Mortality

Duration:

8 d

Dose descriptor:

NOEC

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Fry-Mortality

Duration:

8 d

Dose descriptor:

LOEC

Effect conc.:

7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Fry-Mortality

Key result

Duration:

8 d

Dose descriptor:

LC50

Effect conc.:

6.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

8 d

Dose descriptor:

LC10

Effect conc.:

2.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

8 d

Dose descriptor:

NOEC

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

8 d

Dose descriptor:

LOEC

Effect conc.:

7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

8 d

Dose descriptor:

EC50

Effect conc.:

> 7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

length

Remarks on result:

other: 95% confidence interval: not determined

Duration:

8 d

Dose descriptor:

other: EC20

Effect conc.:

9.019 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

length

Remarks on result:

other: 95% confidence interval: 5.075 - 15.687 mg/L (nominal)

Key result

Duration:

8 d

Dose descriptor:

EC10

Effect conc.:

2.101 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

length

Remarks on result:

other: 95% confidence interval: 1.318 - 3.351 mg/L (nominal)

Key result

Duration:

8 d

Dose descriptor:

NOEC

Effect conc.:

0.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

length

Duration:

8 d

Dose descriptor:

LOEC

Effect conc.:

0.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

length

Details on results:

The difference in the time of hatch occurred randomly and did not show any dose-response relationship. The egg mortality was randomly distributed, no significant toxic effect on this life stage was observed. A clear effect was detectable for the sac fry larvae. Therefore the overall mortality shows nearly the same response relationship as already described for the fry mortality. 86.7% of the control embryos survived u ntil the end of the test. No mortality occurred at the concentrations of 0.3,1.5 and 3.2 mg test item/L. 6.7% overall mortality was observed at a concentration of 0.7 mg test item/L. However, as no mortality was observed in the higher concentrations of 1.5 and 3.2 mg test item/L, this effect was not detected as substance related. A clear substance effect was visible at the highest concentration of 7.0 mg test item/L, where 63.3% of the fish embryos were dead at the end of the test.

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

The LCso. LC» and LCa for length were calculated by using a three parametric normal concentration distribution function (CDF). The NOEC and LOEC were calculated by Williams t-test. a = 0.05, one-sided smaller.

Validity Criteria of the Study

Dissolved Oxygen Concentration: the dissolved oxygen concentration was > 87 % of the air saturation value throughout the test.

Water Temperature: The water temperature was maintained at 25 to 26 °C in the test chambers at any time during the test.

Control Survival: Overall survival of embryos in the negative control was 100 % post hatch until the end of exposure.

Validity criteria fulfilled:

yes

Conclusions:

Based on the test results the LC50 of the test item for Zebrafish (Danio rerio) was determined to be > 7.0 mg test item/L for the egg-and fry-mortality, 6.9 mg test item/L for the overall-mortality and > 7.0 mg test item/L for the parameter length. The NOEC of the test item for zebrafish (Danio rerio) on was determined to be ≥ 7 mg/L for Egg-mortality, 3.2 mg/L for overall mortality and Fry-mortality, all based on nominal concentrations. The EC50 based on body length was found to be > 7 mg/L, the NOEC to be 0.3 mg/L and the EC10 to be 2.101 mg/L (nominal).

Executive summary:

The short-term toxicity of the test item to zebrafish (Danio rerio) was assessed in a Fish Embryo and Sac-fry Stages Test according to according to OECD 212 and GLP principles. For this purpose, fertilised eggs of Zebrafish (30 individuals per treatment) were exposed in a semi-static regime to nominal test item concentrations of 0 (negative control), 7, 3.2, 1.5, 0.7 and 0.3 mg test item/L. The embryos were observed daily for indicators of acute lethality in fish or sublethal effects. The test was terminated just before the yolk-sac of any larvae in any of the test chambers was completely absorbed or before mortalities by starvation started in the controls. The recorded effects were mortality, hatching, growth and symptoms of intoxication of the fish. The NOEC, LOEC and LC50for overall-, egg- and fry-mortality and length endpoints were calculated after 8 d of exposure. The test item concentrations were analytically verified by using liquid chromatography (LC-MS-method) to be 83-85% of the nominal in freshly prepared media, < 80% of the nominal at the start (for the low concentrated samples) and 50-53% of the nominal in the aged media. However, correct dosing of the test item could be demonstrated. Therefore, all biological endpoints were based on nominal test concentrations. All validity criteria of the guideline were met. Based on the test results the LC50 of the test item for Zebrafish (Danio rerio) was determined to be > 7.0 mg test item/L for the egg-and fry-mortality, 6.9 mg test item/L for the overall-mortality and > 7.0 mg test item/L for the parameter length. The NOECs of the test item for zebrafish (Danio rerio) was determined to be ≥ 7 mg/L for Egg-mortality, 3.2 mg/L for overall mortality and Fry-mortality in nominal. Based on body length sub-lethal effects were determined: the EC50 was found to be > 7 mg/L, the NOEC to be 0.3 mg/L and the EC10 to be 2.101 mg/L (nominal).

Description of key information

Based on the test results the LC50 of the test item for Zebrafish (Danio rerio) was determined to be > 7.0 mg test item/L for the egg-and fry-mortality, 6.9 mg test item/L for the overall-mortality and > 7.0 mg test item/L for the parameter length. The NOEC of the test item for zebrafish (Danio rerio) on was determined to be ≥ 7 mg/L for Egg-mortality, 3.2 mg/L for overall mortality and Fry-mortality, all based on nominal concentrations. The EC50 based on body length was found to be > 7 mg/L, the NOEC to be 0.3 mg/L and the EC10 to be 2.101 mg/L (nominal).

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

2.101 mg/L

Additional information

The short-term toxicity of the test item to zebrafish (Danio rerio) was assessed in a Fish Embryo and Sac-fry Stages Test according to according to OECD 212 and GLP principles. For this purpose, fertilised eggs of Zebrafish (30 individuals per treatment) were exposed in a semi-static regime to nominal test item concentrations of 0 (negative control), 7, 3.2, 1.5, 0.7 and 0.3 mg test item/L. The embryos were observed daily for indicators of acute lethality in fish or sublethal effects. The test was terminated just before the yolk-sac of any larvae in any of the test chambers was completely absorbed or before mortalities by starvation started in the controls. The recorded effects were mortality, hatching, growth and symptoms of intoxication of the fish. The NOEC, LOEC and LC50 for overall-, egg- and fry-mortality and length endpoints were calculated after 8 d of exposure. The test item concentrations were analytically verified by using liquid chromatography (LC-MS-method) to be 83-85% of the nominal in freshly prepared media, < 80% of the nominal at the start (for the low concentrated samples) and 50-53% of the nominal in the aged media. However, correct dosing of the test item could be demonstrated. Therefore, all biological endpoints were based on nominal test concentrations. All validity criteria of the guideline were met. Based on the test results the LC50 of the test item for Zebrafish (Danio rerio) was determined to be > 7.0 mg test item/L for the egg-and fry-mortality, 6.9 mg test item/L for the overall-mortality and > 7.0 mg test item/L for the parameter length. The NOECs of the test item for zebrafish (Danio rerio) was determined to be ≥ 7 mg/L for Egg-mortality, 3.2 mg/L for overall mortality and Fry-mortality in nominal. Based on body length, sub-lethal effects were determined: the EC50 was found to be > 7 mg/L, the NOEC to be 0.3 mg/L and the EC10 to be 2.101 mg/L (nominal).

The reported effect concentrations refer to the commercial formulation containing 46.2% (w/w) of the REACH registration substance which reflects the test item used in the study.