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Short-term toxicity to aquatic invertebrates

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Reference
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 February 2018 to 18 March 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
no
Principles of method if other than guideline:
NA
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
Samples were taken from the control and each test group from the bulk test preparation at 0 hours and from the pooled replicates (R1 to R4) at 48 hours for quantitative analysis. Samples were stored frozen prior to analysis. Duplicate samples were taken and stored frozen for further analysis if necessary.
Vehicle:
no
Details on test solutions:
Due to the low aqueous solubility and complex nature of the test item, the test medium was prepared as a WAF of the test item.

Range-finding test:
The loading rates used in the definitive test were determined by a preliminary range-finding test, in which Daphnia magna were exposed to a series of nominal loading rates of 1.0, 10 and 100 mg/L. A sample of each loading rate WAF was taken for chemical analysis at 0 and 48 hours in order to determine the stability of the test item under test conditions. All samples were stored frozen prior to analysis. Only concentrations within the range to be used for the definitive test were analysed.

After 48 hours exposure, no immobilisation was observed in the control and at 1.0 mg/L loading rate WAF, however 100% immobilisation was observed at the 10 and 100 mg/L loading rate WAF. Sub-lethal effects of exposure were observed in the 10 mg/L loading rate WAF at 24 hours. This response was reduced mobility. Based on this information loading rates of 1.0, 1.8, 3.2, 5.6 and 10 mg/L were selected for the definitive test.


Definitive test:
Based on the results of the range-finding test the following loading rates were assigned to the definitive test: 1.0, 1.8, 3.2, 5.6 and 10 mg/L.

Experimental preparation:
Nominal amounts of test item (5.0, 9.0, 16, 28 and 50 mg) were each separately added to the surface of 5 litres of test water to give the 1.0, 1.8, 3.2, 5.6 and 10 mg/L loading rates respectively. After the addition of the test item, the test water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The vortex depth was recorded at the start and end of the mixing period. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1 hour. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75 to 100 mL discarded) to give the 1.0, 1.8, 3.2, 5.6 and 10 mg/L loading rate WAFs. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present.

The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 48 hours.
Test organisms (species):
Daphnia magna
Details on test organisms:
The test was carried out using first instar Daphnia magna derived from in-house laboratory cultures.

Adult daphnids were maintained in 150 mL glass beakers containing 100 mL Elendt M7 medium in a temperature controlled room maintaining the water temperature at 18 to 22°C. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods. Each culture was fed daily with a mixture of algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension. Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Remarks on exposure duration:
No remarks
Post exposure observation period:
NA
Hardness:
NA
Test temperature:
20 to 22 ºC
pH:
7.8 to 7.9
Dissolved oxygen:
8.0 to 8.6 mg O2/L
Salinity:
NA
Conductivity:
NA
Nominal and measured concentrations:
Nominal loading rates: 1.0, 1.8, 3.2, 5.6 and 10 mg/L
Details on test conditions:
Exposure Conditions:
Test vessels were 150 mL glass jars containing approximately 100 mL of test preparation. At the start of the test five daphnids were placed in each test and control vessel at random, in the test preparations. Four replicate test and control vessels were prepared (the range-finding test included four replicate vessels for the control and 100 mg/L loading rate and two replicate vessels for the 1.0 and 10 mg/L loading rates). The test vessels were then covered to reduce evaporation and maintained in a temperature controlled room maintaining the water temperature at 18 to 22 °C with a maximum deviation of ±1 °C with a photoperiod of 16 hours light (between 200 and 1200 lux) and 8 hours darkness with 20 minute dawn and dusk transition periods. The daphnids were not individually identified, received no food during exposure and the test vessels were not aerated. The control group was maintained under identical conditions but not exposed to the test item. The test preparations were not renewed during the exposure period.


Test condition measurements:
Water temperature was recorded daily throughout the test. Dissolved oxygen concentrations and pH were recorded at the start and termination of the test. The pH and dissolved oxygen concentration were measured using a Hach Flexi handheld meter whilst the temperature was measured using a Hanna Instruments HI 93510 digital thermometer. The light intensity during the light period was measured using an ATP Instrumentation Lux meter. The appearance of the test media was recorded daily. The vortex depth was recorded at the start and end of the mixing period.


Test Organism Observations:
Any immobilisation or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. The criterion of effect used was that daphnia were considered to be immobilised if they were unable to swim within 15 seconds after gentle agitation.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
24 h
Dose descriptor:
EL50
Effect conc.:
9.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
mobility
Remarks on result:
other: 95% Confidence Limits not determined
Key result
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
6.9 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
mobility
Remarks on result:
other: 95% Confidence Limits not determined
Duration:
48 h
Dose descriptor:
NOELR
Effect conc.:
5.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
mobility
Remarks on result:
other: 95% Confidence Limits not determined
Duration:
48 h
Dose descriptor:
LOELR
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
mobility
Remarks on result:
other: 95% Confidence Limits not determined
Details on results:
Chemical analysis of the fresh test preparations at 0 hours showed measured test concentrations to range from 2.7 to 9.3 mg/L. Chemical analysis of the aged test preparations at 48 hours showed measured test concentrations to range from 2.5 to 8.6 mg/L. The dissolved test item may have been one or several components of the test item. Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test item as a whole, the results were based on nominal loading rates only.

No sub-lethal effects of exposure were observed throughout the test in the control and at loading rates of 1.0, 1.8, 3.2 and 5.6 mg/L, however, a sub-lethal effect of exposure was observed in the 10 mg/L loading rate at 24 hours. This response was reduced mobility.
Results with reference substance (positive control):
The positive control used potassium dichromate as the reference item at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L and exposure conditions similar to those in the definitive test.

The results from the positive control with potassium dichromate were 24-hour EC50, NOEC and LOEC values of 0.79, 0.56 and 1.0 mg/L, respectively and 48-hour EC50, NOEC and LOEC values of 0.75, 0.56 and 1.0 mg/L, respectively. Results were within the normal range for this reference item (i.e. 24-hour EC50 for potassium dichromate within the range 0.6 mg/L to 2.1 mg/L, as per the results of the inter laboratory tests and a Technical Corrigendum to ISO 6341).
Reported statistics and error estimates:
The EL50 values and associated confidence limits at 24 and 48 hours and the slope of the response curve and its standard error were calculated by Logit analysis using Linear Maximum-Likelihood regression. The Lowest Observed Effect Loading (LOEL) rate and the NOEL rate at 24 and 48 hours were calculated using the Step-down Cochran Armitage Test Procedure. All results were calculated using the ToxRat Professional computer software package (TOXRAT).

Cumulative Immobilization Data and Observations in the Range-finding Test

Nominal Loading Rate (mg/L)

24 hours

Cumulative immobilised Daphnia (initial population : 5 per replicate)

Observations

Rep1

Rep2

Rep3

Rep4

Total

%

Rep1

Rep2

Rep3

Rep4

Control

0

0

0

0

0

0

5 N

5 N

5 N

5 N

1

0

0

-

-

0

0

5 N

5 N

-

-

10

0

1

-

-

1

10

2 R 3 N

1 R 3 N

-

-

100

5

5

5

5

20

100

AI

AI

AI

AI

48 hours

Control

0

0

0

0

0

0

5 N

5 N

5 N

5 N

1

0

0

-

-

0

0

5 N

5 N

-

-

10

5

5

-

-

10

100

AI

AI

-

-

100

5

5

5

5

20

100

AI

AI

AI

AI

 

Rep = Replicate

N = Normal

R = Reduced mobility

AI = All daphnia immobilized

- = Not applicable

Cumulative Immobilization Data and Observations in the Definitive Test

  

Nominal Loading Rate (mg/L)

24 hours

Cumulative immobilised Daphnia (initial population : 5 per replicate)

Observations

Rep1

Rep2

Rep3

Rep4

Total

%

Rep1

Rep2

Rep3

Rep4

Control

0

0

0

0

0

0

5 N

5 N

5 N

5 N

1

0

0

0

0

0

0

5 N

5 N

5 N

5 N

1.8

0

0

0

1

1

5

5 N

5 N

5 N

4 N

3.2

0

0

0

0

0

0

5 N

5 N

5 N

5 N

5.6

0

1

0

0

1

5

5 N

4 N

5 N

5 N

10

3

3

4

3

13

65

1 R 1 N

2 N

1 N

2 N

48 hours

Control

0

0

0

0

0

0

5 N

5 N

5 N

5 N

1

0

0

0

0

0

0

5 N

5 N

5 N

5 N

1.8

0

1

0

1

2

10

5 N

4 N

5 N

4 N

3.2

0

0

0

0

0

0

5 N

5 N

5 N

5 N

5.6

0

1

0

0

1

5

5 N

4 N

5 N

5 N

10

5

5

5

5

20

100

AI

AI

AI

AI

  

Rep = Replicate

N = Normal

R = Reduced mobility

AI = All daphnia immobilized

- = Not applicable

Validity criteria fulfilled:
yes
Conclusions:
The acute toxicity of the test item 'Reaction product of 2,3-epoxypropyl neodecanoate and Benzenesulfonic acid, C10-13-sec-alkyl derivatives' to Daphnia magna resulted in a 48-hour EL50 value of 6.9 mg/L.
Executive summary:

The acute toxicity of 'Reaction product of 2,3-epoxypropyl neodecanoate and Benzenesulfonic acid, C10-13-sec-alkyl derivatives' to the freshwater invertebrate Daphnia magna was assessed according to the OECD Guideline 202 ("Daphnia sp., acute immobilisation test" eferenced as Method C.2 of Commission Regulation (EC) No. 440/2008). The study was conducted at a GLP accredited laboratory and met the validity criteria described in the Guideline and is therefore considered to be reliable.

 

Due to the low aqueous solubility and complex nature of the test item, the test medium was prepared as a Water Accommodated Fraction (WAF). Following a preliminary range-finding test, 20 daphnids (four replicates of five animals) were exposed to WAFs of the test item over a range of nominal loading rates of 1.0, 1.8, 3.2, 5.6 and 10 mg/L for 48 hours at a temperature of approximately 20 to 22°C under static test conditions. The number of immobilised daphnids and any adverse reactions to exposure were recorded after 24 and 48 hours.

 

Chemical analysis of the fresh test preparations at 0 hours showed measured test concentrations to range from 2.3 to 9.3 mg/L. Chemical analysis of the aged test preparations at 48 hours showed measured test concentrations to range from 2.5 to 8.6 mg/L. The dissolved test item may have been one or several components of the test item. Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test item as a whole, the results were based on nominal loading rates only. The study resulted in 48-hour EL50, NOELR and LOELR values of 6.9, 5.6 and 10 mg/L, respectively.

Description of key information

The acute toxicity of 'Reaction product of 2,3-epoxypropyl neodecanoate and Benzenesulfonic acid, C10-13-sec-alkyl derivatives' to the freshwater invertebrate Daphnia magna was assessed according to the OECD Guideline 202 ("Daphnia sp., acute immobilisation test" referenced as Method C.2 of Commission Regulation (EC) No. 440/2008). The study was conducted at a GLP accredited laboratory and met the validity criteria described in the Guideline and is therefore considered to be reliable.

Due to the low aqueous solubility and complex nature of the test item, the test medium was prepared as a Water Accommodated Fraction (WAF). Following a preliminary range-finding test, 20 daphnids (four replicates of five animals) were exposed to WAFs of the test item over a range of nominal loading rates of 1.0, 1.8, 3.2, 5.6 and 10 mg/L for 48 hours at a temperature of approximately 20 to 22°C under static test conditions. The number of immobilised daphnids and any adverse reactions to exposure were recorded after 24 and 48 hours.

The study resulted in 48-hour EL50, NOELR and LOELR values of 6.9, 5.6 and 10 mg/L, respectively.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
6.9 mg/L

Additional information

Chemical analysis of the fresh test preparations at 0 hours showed measured test concentrations to range from 2.3 to 9.3 mg/L. Chemical analysis of the aged test preparations at 48 hours showed measured test concentrations to range from 2.5 to 8.6 mg/L. The dissolved test item may have been one or several components of the test item. Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test item as a whole, the results were based on nominal loading rates only.