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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:
21 February 2017 to 24 February 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
10 January 2017
Analytical monitoring:
yes
Details on sampling:
- Chemical analyses: Single samples for analysis were taken from the control and all test concentrations.
- Frequency of sampling: At the start (t=0h) and the end of the test (t=48h).
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- The mixing vessel was a cylindrical glass bottle sealed with a screw cap and fitted with a drain port near the bottom for drawing off the saturated stock solution. The volume of the mixing vessel was approximately 1 L. A magnetic stirring bar was placed in the vessel and approx. 1 L test water was added. Then an excess of the test item (approx. 4 g) was carefully added directly to the surface of the test water, and nitrogen was injected into the headspace in order to prevent potential polymerisation of the test item in test water (as observed during pre-experiments). Then the mixing vessel was sealed and the mixing was initiated with the vortex in the centre extending maximally around 10% vessel depth from the top to the bottom of the vessel. The stirring speed was as low as possible to maintain mixing of the water phase without dispersing the test substance in the water phase. After 7 days and +/- 2 hours of gentle stirring in the dark and at room temperature, the saturated aqueous phase was extracted via the drain port. The first 100 mL were removed and samples were taken from the remaining stock solution and chemically analysed. Then the stock solution was diluted with test water as necessary into 200-mL volumetric flasks to obtain the required test concentrations based on the measured concentration of the stock solution (ca. 5-6 mg.L-1). Each prepared concentration was inverted several times before filling the test tube (without headspace) to ensure adequate mixing and homogeneity. After filling and introduction of daphnids the vessels were sealed immediately. The test was carried out without adjustment of the pH.

- Controls: Test water without test substance but treated in the same way as the test substance solutions.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Strain: Daphnia magna (Straus), clone 5
- Source: LIEBE - CNRS UMR 7146 - UFR SciFA - Université de Lorraine Campus Bridoux - Bât. IBISE, 8, rue du Général Delestraint - 57070 METZ, bred in the Laboratoires des Pyrénées et des Landes.
- Daphnids originated from a healthy stock, showing no signs of stress such as mortality, presence of males, ephippia or discoloured animals.
- Age at study initiation: < 24 h
- Breeding Conditions: Daphnids were cultured in the Laboratoires des Pyrénées et des Landes under similar temperature and light conditions as used in the test. The cultivation of the parental daphnids was performed in all-glass vessel containing test water. Cultures were maintained at a density of 1 adult daphnid per 25 mL of culture medium. Daphnids were fed at least three times a week with a suspension of algal cells (Pseudokirchneriella subcapitata) up to 0.1-0.2 mg C.Daphnia.-1day.-1. The water was changed three times a week. These culture conditions maintained the daphnids in the parthenogenetic reproductive stage.
- Feeding during test: No feeding
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Remarks on exposure duration:
None
Post exposure observation period:
None
Hardness:
Total water hardness was approximately 250 mg/L (as CaCO3)
Test temperature:
Between 20.8 and 20.9 °C throughout the test (average value: 20.9°C)
pH:
7.90 - 8.55
requirement: pH: 6.0-9.0, not varying by more than 1.5 units
Dissolved oxygen:
5.15 - 8.46 mg O2/L
Dissolved oxygen concentration at the end of the test was ≥ 60% of the air-saturation value in controls and test vessels, except at 3.17 mg.L-1 and 5.86 mg.L-1, suggesting that pH and oxygen concentrations were not influenced by the test item.
Salinity:
No data
Conductivity:
No data
Nominal and measured concentrations:
Nominal: 0.50; 0.93; 1.71; 3.17; 5.86 mg test item.L-1
Corresponding average exposure concentrations (geometric means): 0.33; 0.43; 0.35; 0.21; 0.21 mg.L-1
Details on test conditions:
TEST SYSTEM
- Test vessel: 60-mL glass flasks sealed with assembled screw cap with hole and PTFE/silicone septum. Each test vessel was uniquely identified with study code, replicate number, date of experimentation and concentration.
- Aeration: No aeration of the test solutions occurred throughout the test.
- Test type: static
- No. of daphnids: 20 per control and test concentration, divided into 4 groups of 5 animals
- Loading: 5 daphnids per vessel each completely filled with test solution and without headspace
- Introduction of Daphnids: Daphnids were introduced into the test vessel each completely filled with test solution and without headspace immediately after filling the test vessels with test solutions.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water as prescribed by the OECD Guideline 202.

OTHER TEST CONDITIONS
- Light regime: 16 h light : 8 h darkness

EFFECT PARAMETERS MEASURED:
- Immobility: Eventual immobility and abnormal behaviour were determined by visual observation after 24 and 48 hours. Daphnids were considered to be immobile if they were not able to swim within 15 seconds after gentle agitation of test vessels.
- pH and dissolved O2: At the beginning and at the end of the test from all test concentrations and control.
- Temperature of Medium: Measured continuously in a temperature controlled vessel next to the test vessels, over the study period, beginning at the start of the test.
Test conditions were recorded with suitable instruments and documented in the raw data.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 0.33 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
After 48 hours of exposure, immobilisations were 15% at 0.33 mg.L-1, and 100% in the other test concentrations. Due to the severe test item losses observed in the highest test concentrations, their geometric means gave average exposure concentrations lower than the lowest tested concentrations, thus it was not possible to determine an EC50 with statistical tools. However, it was decided to provide a 48-hour EC50 directly based from the raw data, i.e. > 0.33 mg.L-1 and < 1.00 mg.L-1, in agreement with the Study Monitor.
The 48-hour EC50 value was estimated to be higher than 0.33 mg/ test item.L-1.
Results with reference substance (positive control):
On April 4, 2017 (most recent test), the 24h-EC50 was 0.97 mg.L-1. Hence, the sensitivity of the clone of Daphnia magna was in agreement with OECD 202 (1) (expected 24h-EC50: 0.6 mg.L-1 to 2.1 mg.L-1) at this time.
Reported statistics and error estimates:
No statistical analysis was performed. Effective concentrations were determined directly from the raw data.

Table 6.1.3/1: Acute immobilisation of daphnids after 24 and 48 h in the final test

 

Nominal concentration(mg test item.L-1)

Replicate

Number of daphnids exposed

Response at 24h

Response at 48h

Number

Total %

Number

Total %

Control

1

2

3

4

5

5

5

5

0

0

0

0

0

0

0

0

0

0

0.50

1

2

3

4

5

5

5

5

0

0

0

0

0

0

1

1

1

15

0.93

1

2

3

4

5

5

5

5

1

0

1

0

10

5

5

5

5

100

1.71

1

2

3

4

5

5

5

5

1

1

3

4

45

5

5

5

5

100

3.17

1

2

3

4

5

5

5

5

0

0

0

0

0

5

5

5

5

100

5.86

(max sol.)

1

2

3

4

5

5

5

5

5

5

5

5

100

5

5

5

5

100

Water quality parameters and environmental conditions throughout the test:

The results of measurement of pH and oxygen concentrations (mg.L-1) remained within the limits prescribed by the study plan (pH: 6.0-9.0, not varying by more than 1.5 units; oxygen:≥ 60% of the air-saturation valueat the end of the test, except at 3.17mg.L-1and 5.86 mg.L-1, suggesting that pH and oxygen concentrations were not influenced by the test item.

 

Validity criteria of the study:

Controls: In the control, no daphnids became immobilised nor trapped at the surface of the water nor showed signs of stress.

Dissolved [O2]: The dissolved oxygen concentration was at least 60% of the air saturation value throughout the test in all test vessels (see Table 3 - Appendix II), except at the nominal test concentrations of 3.17 and 5.86 mg.L-1where dissolved [O2] were below 60 % of the air saturation value. It was assumed that these decreases (probably due to an unavoidable reaction of the test item with O2contained in the test water) were not the cause of the observed immobilisations, since [O2] values were in line with [O2] tolerance range for daphnids (as indicated in OECD Guideline 202, where the dissolved oxygen concentration at the end of the test should be higher than 3 mg.L-1in control and test vessels). Besides, it should be noted that 100% immobilisation was also recorded at the nominal test concentrations of 0.93 and 1.71 mg.L-1(see 3.4. for details) within 48 hours and [O2] concentrations were well higher than 60% of the air-saturation value, which supports the assumption stated above. This deviation was therefore not considered to have affected the results and the integrity of the study, in agreement with the Study Monitor.

Validity criteria fulfilled:
yes
Conclusions:
The toxic effect of test item (E,Z)-2,6-DIMETHYLOCTA-2,4,6-TRIENE to freshwater invertebrate Daphnia magna was investigated in a closed semi-static test. Under the experimental conditions and based on the average of measured exposure concentrations, the 48-hour EC50 value was estimated to be higher than 0.33 mg test item.L-1. Therefore, the substance is classified in category 1 for acute toxicity and for long-term toxicity according to CLP regulation (EC) No 1272/2008 and GHS regulation. Hazard sentences H400 and H410 are required as well as signal word Danger.
Executive summary:

A study was performed to assess the acute toxicity of test item (E,Z)-2,6-DIMETHYLOCTA-2,4,6-TRIENE to Daphnia magna. The method followed was designed to be compliant with OECD Guideline for Testing of Chemicals No. 202, “Daphnia sp., Acute Immobilisation Test”, referenced as Method C.2 of Commission Regulation No. 440/2008 (amended by Commission Regulation (EU) 2016/266) and with the “Guidance document on aquatic toxicity testing of difficult substances and mixtures” (OECD No. 23).

Twenty daphnids (four replicates, five daphnids per replicate) were exposed to an aqueous solution of the test item over a range of nominal test concentrations of 0.50, 0.93, 1.71, 3.17 and 5.86 mg test item.L-1(concentrationclose to the solubility limit of the test item in the test water) and to a control.The immobility of the daphnids was determined in a semi-static 48-hour test by visual observation after 24 and 48 hours. The concentrations of the test item were determined by chemical analyses at the start (t=0h), at t=24h (new and old solutions) and at the end of the test (t=48h).

Although every effort was made to maintain the concentrations of the test item in the test vessels (nitrogen injection into the headspace during stock solution preparation, renewal of the test solutions at t=24h and closed conditions without headspace), chemical analyses revealed severe test item losses that were proportional to the tested concentrations.Since the deviation of the exposure concentrations of the test substance was greater than 20% of the initial concentrations, the results were expressed in terms of geometric means of the exposure concentrations for the present study.

After 48 hours of exposure, immobilisations were 15% at 0.33mg.L-1,and 100% in the other test concentrations.Due to the severe test item losses observed in the highest test concentrations, their geometric means gave average exposure concentrations lower than the lowest tested concentrations, thus it was not possible to determine an EC50with statistical tools. However, it was decided to provide a 48-hour EC50directly based from the raw data, i.e. > 0.33mg.L-, in agreement with the Study Monitor.

The toxic effect of test item (E,Z)-2,6-DIMETHYLOCTA-2,4,6-TRIENE to the freshwater invertebrateDaphnia magna was investigated in a closed semi-static test. Under the experimental conditions and based on the average of measured exposure concentrations, the 48-hour EC50value was estimated to be higher than 0.33 mg test item.L-1.

Therefore, the substance is classified in category 1 for acute toxicity and for long-term toxicity according to CLP regulation (EC) No 1272/2008 and GHS regulation. Hazard sentences H400 and H410 are required as well as signal word Danger.

Description of key information

(E,Z)-2,6 -dimethylocta-2,4,6 -triene exhibits a 48h-EC50 for freshwater invertebrates higher than 0.33 mg test item.L-1.

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
0.33 mg/L

Additional information

One reliable study is available for the registered substance. The toxicity to freshwater invertebrates was investigated in a static test, according to Guideline OECD 202, referenced as Method C.2 of Commission Regulation No. 440/2008, and under GLP compliance. Daphnids (Daphnia magna) were exposed to an aqueous solution of the test item at nominal test concentrations of 0.50, 0.93, 1.71, 3.17, and 5.86 mg/L and to a control, over a test period of 48 hours (semi-static conditions). Samples taken from the control and all test concentrations were analysed at the start, at t=24 h (new and old solutions) and at the end of the test (t=48 h) in order to determine maintenance of actual concentrations of the test item.

After 48 hours of exposure, immobilisations were 15% at 0.33mg.L-1,and 100% in the other test concentrations. Due to the severe test item losses observed in the highest test concentrations, their geometric means gave average exposure concentrations lower than the lowest tested concentrations, thus it was not possible to determine an EC50with statistical tools.

However, it was decided to provide a 48-hour EC50 directly based from the raw data, i.e. > 0.33 mg.L-1, in agreement with the Study Monitor.

Therefore, the substance is classified in category 1 for acute toxicity and for long-term toxicity according to CLP regulation (EC) No 1272/2008 and GHS regulation. Hazard sentences H400 and H410 are required as well as signal word Danger.