Registration Dossier

Ecotoxicological information

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 27 July 2015 and 28 September 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

Test guidelineopen allclose all
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
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
(3S,6E)-3,7,11-trimethyldodeca-6,10-dienal
EC Number:
810-298-1
Cas Number:
194934-66-2
Molecular formula:
C15H26O
IUPAC Name:
(3S,6E)-3,7,11-trimethyldodeca-6,10-dienal
Test material form:
liquid

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Verification of Test Concentrations
Samples were taken from the control and each test group from the bulk fresh test preparation at 0 and 24 hours and from the pooled aged replicates at 24 and 48 hours for quantitative analysis. All samples were stored frozen prior to analysis. Duplicate samples were taken at 0, 24 and 48 hours and stored frozen for further analysis if necessary.

Test solutions

Vehicle:
no
Details on test solutions:
Range-finding Test
A nominal amount of test item (1100 mg) was dispersed in 11 liters of test water with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 µm Sartorius Sartopore (first approximate 2 liters discarded in order to pre-condition the filter) to give a 100% v/v saturated solution. A series of dilutions was made from this saturated solution to give further test concentrations of 0.10, 1.0 and 10% v/v saturated solution.
Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.


Definitive Test
A nominal amount of test item (1100 mg) was dispersed in 11 liters of test water with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 µm Sartorius Sartopore filter (first approximate 2 liters discarded in order to pre-condition the filter) to give a 100% v/v saturated solution. A series of dilutions was made from this saturated solution to give further test concentrations of 2.6, 6.4, 16 and 40% v/v saturated solution.

Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
The test was carried out using 1st instar Daphnia magna derived from in-house laboratory cultures.

Adult daphnia were maintained in 150 mL glass beakers containing Elendt M7 medium in a temperature controlled room at approximately 20 °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.

Study design

Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h

Test conditions

Test temperature:
The water temperature was recorded daily throughout the test. The measurements at 0 hours and after the test media renewal at 24 hours represent those of the freshly prepared test preparations while the measurements taken prior to the test media renewal, and on termination of the test after 48 hours, represent those of the used or 24-Hour old test preparations. The temperature was measured using a Hanna Instruments HI 93510 digital thermometer. Temperature was maintained at approximately 21°C throughout the test.
pH:
The pH was recorded daily throughout the test. The measurements at 0 hours and after the test media renewal at 24 hours represent those of the freshly prepared test preparations while the measurements taken prior to the test media renewal, and on termination of the test after 48 hours, represent those of the used or 24-Hour old test preparations. The pH was measured using a Hach Flexi handheld meter whilst the temperature was measured using a Hanna Instruments HI 93510 digital thermometer. There were no treatment related differences for pH.
Dissolved oxygen:
The dissolved oxygen concentrations were recorded daily throughout the test. The measurements at 0 hours and after the test media renewal at 24 hours represent those of the freshly prepared test preparations while the measurements taken prior to the test media renewal, and on termination of the test after 48 hours, represent those of the used or 24-Hour old test preparations. The dissolved oxygen concentration was measured using a Hach Flexi handheld meter. There were no treatment related differences for oxygen concentration.
Nominal and measured concentrations:
Range-finding Test: Nominal test concentrations of 0.10, 1.0, 10 and 100% v/v saturated solution.
Definitive Test: Nominal test concentrations of 2.6, 6.4, 16 and 40% v/v saturated solution.
Details on test conditions:
Test Water
Reconstituted water (ISO medium) was used for both the range-finding and definitive.

Procedure
Preliminary Media Preparation Trial
Preliminary solubility work conducted indicated that the test item was practically insoluble in water using traditional methods of preparation e.g. ultrasonication and high shear mixing.

Based on this information the test item was categorized as being a ‘difficult substance’ as defined by the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 2000). Therefore a media preparation trial was conducted in order to determine the solubility of the test item under test conditions.


Range-finding Test
The results obtained from the preliminary media preparation trial conducted indicated that a dissolved test item concentration of approximately 1.35 mg/L could be obtained using a saturated solution method of preparation.

The test concentrations to be used in the definitive test were determined by a preliminary range-finding test.

In the range-finding test Daphnia magna were exposed to a series of nominal test concentrations of 0.10, 1.0, 10 and 100% v/v saturated solution.

A nominal amount of test item (1100 mg) was dispersed in 11 liters of test water with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 µm Sartorius Sartopore (first approximate 2 liters discarded in order to pre-condition the filter) to give a
100% v/v saturated solution. A series of dilutions was made from this saturated solution to give further test concentrations of 0.10, 1.0 and 10% v/v saturated solution.

Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

In the range-finding test 10 daphnids were placed in each test and control vessel and maintained in a temperature controlled room at approximately 22 °C with a photoperiod of 16 hours light and 8 hours darkness for a period of 48 hours with 20 minute dawn and dusk transition periods. Each 150 mL test and control vessel contained 100 mL of test media and was covered to reduce evaporation. After 24 and 48 hours the number of immobilized daphnids were recorded.

The control group was maintained under identical conditions but not exposed to the test item.

A sample of each test concentration 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 analyzed.


Definitive Test
Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 2.6, 6.4, 16, 40 and 100% v/v saturated solution.

Experimental Preparation
A nominal amount of test item (1100 mg) was dispersed in 11 liters of test water with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 µm Sartorius Sartopore filter (first approximate 2 liters discarded in order to pre-condition the filter) to give a 100% v/v saturated solution. A series of dilutions was made from this saturated solution to give further test concentrations of 2.6, 6.4, 16 and 40% v/v saturated solution.

Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0, 24 and 48 hours (see Appendix 6).


Exposure Conditions
As in the range-finding test 150 mL glass beakers containing approximately 100 mL of test preparation were used. At the start of the test 5 daphnids were placed in each test and control vessel at random, in the test preparations. Four replicate test and control vessels were prepared. The test vessels were then covered to reduce evaporation and maintained in a temperature controlled room at approximately 21°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.

Semi-static test conditions were employed in the test in an effort to maintain dissolved test item concentrations. For the test media renewal at 24 hours, the test concentrations were freshly prepared and the daphnids transferred by wide bore pipette from the 24-Hour old test media into the fresh test media.


Evaluations
Test Organism Observations
Any immobilization 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 immobilized if they were unable to swim within 15 seconds after gentle agitation.
Reference substance (positive control):
yes
Remarks:
potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
0.36 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
0.29 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
LOEC
Effect conc.:
0.74 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
Range-finding Test
No immobilization was observed at the test concentrations of 0.10, 1.0 and 10% v/v saturated solution, however, immobilization was observed at 100% v/v saturated solution.

Based on this information test concentrations of 2.6, 6.4, 16, 40 and 100% v/v saturated solution were selected for the definitive test.

Chemical analysis of the test preparations at 0 hours showed measured test concentrations to range from less than the limit of quantification (LOQ), determined to be 0.010 mg/L to 1.05 mg/L. There was a significant decline in the measured concentrations at 48 hours in the range of less than the LOQ to 0.293 mg/L indicating that the test item was not stable under test conditions.


Definitive Test
Immobilization Data
Analysis of the immobilization data by Probit analysis using Linear Maximum-Likelihood regression at 24 and 48 hours based on the geometric mean measured test concentrations gave the following results:

The 24 h EC50 was 0.74 mg/L. 95 % confidence limits could not be determined, as data did not fit available models.
The 48 h EC50 was 0.36 mg/L. 95 % confidence limits could not be determined, as data did not fit available models.

The No Observed Effect Concentration after 24 and 48 hours exposure was 0.29 mg/L. The Lowest Observed Effect Concentration after 24 and 48 hours exposure was 0.74 mg/L.

The slopes and their standard errors of the response curves at 24 and 48 hours were 2.52 (SE = 0.11) and 1.57 (SE = 0.12) respectively.


Sub-Lethal Effects
Sub-lethal effects of exposure were observed in the 6.4, 16 and 40% v/v saturated solutions after 48 hours. The response observed was trapping at the surface and was believed to be attributable to exposure to the test item.
Results with reference substance (positive control):
A positive control (Study Number 41500252) used potassium dichromate as the reference item at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L.
Exposure conditions for the positive control were similar to those in the definitive test.

Analysis of the immobilization data by probit analysis using the linear maximum likelihood regression method at 24 and 48 hours using the ToxRat Professional computer software package based on the nominal test concentrations gave the following results:

The 24 h EC50 was 1.1 mg/L. It was not possible to determine 95 % confidence limits.
The 48 h EC50 was 0.75 mg/L with 95% confidence limits of 0.69 - 0.82 mg/L.
The NOEC was 0.56 mg/L and the LOEC was 1.0 mg/L.
The No Observed Effect Concentration is based upon equal to or less than 10% immobilization at this concentration.
The results from the positive control with potassium dichromate were within the normal range for this reference item.
Reported statistics and error estimates:
Statistical Analysis
The EC50 values and associated confidence limits at 24 and 48 hours and the slope of the response curve and its standard error were calculated by Probit analysis using Linear Maximum-Likelihood regression. The Lowest Observed Effect Concentration and the No Observed Effect Concentration at 24 and 48 hours were calculated using the Fisher’s Exact Binomial Test with Bonferroni correction. All results were calculated using the ToxRat Professional computer software package (TOXRAT).


Geometric Mean Measured Test Concentrations
The geometric mean measured test concentrations of the samples were calculated as follows using the measured test concentrations of replicates R1 – R4 pooled:

GM = √(C0C1)

Where:

GM = geometric mean measured test concentration (mg/L)
C0 = measured concentration at the start of the test (mg/L)
C1 = measured concentration at the end of the test (mg/L)

The geometric mean measured concentration was determined for each exposure period (0-Hour fresh to 24-Hour old and 24-Hour fresh to 48-Hour old) and the arithmetic average calculated of these values.

Any other information on results incl. tables

Verification of Test Concentrations

Analysis of the freshly prepared test media at 0 and 24 hours showed measured test concentrations to range from 0.014 to 1.30 mg/L. Analysis of the old test preparations at 24 and 48 hours showed measured test concentrations had declined, to range from less than the LOQ (0.010 mg/L) to 0.49 mg/L and hence it was considered appropriate to calculate the results based on the geometric mean measured test concentration in order to give a “worst case” analysis of the data.

 

The geometric mean measured test concentrations were determined to be:

 

Nominal Test Concentration
(% v/v Saturated Solution)

Geometric Mean Measured Test Concentration (mg/L)

Expressed as a % of the 0-Hour Measured Test Concentration

2.6

0.010

71

6.4

0.018

25

16

0.080

45

40

0.29

63

100

0.74

70

 

Immobilization Data

Analysis of the immobilization data by Probit analysis using Linear Maximum-Likelihood regression at 24 and 48 hours based on the geometric mean measured test concentrations gave the following results:

 

Time (h)

EC50(mg/L)

95% Confidence limits
(mg/L)

24

0.74

n.d.

48

0.36

n.d.


n.d. = not determinedas data did not fit available models

Validation Criteria

The test was considered to be valid given that no more than 10% of the control daphnids showed immobilization or other signs of disease or stress and that the oxygen concentration at the end of the test was equal to or greater than 3 mg/L in the control and test vessels.

Observations on Test Item Solubility

At the start and throughout the test all control and test solutions were observed to be clear colorless solutions.

Cumulative Immobilization Data and Observations in the Range-finding Test

Nominal
Concentration
(% v/v Saturated Solution)

Observations (Initial Population: 10 Per Replicate)

24 Hours

48 Hours

Cumulative Immobilized Daphnia

Observations

Cumulative Immobilized Daphnia

Observations

Control

0

10 N

0

10 N

0.10

0

10 N

0

9 N 1 T

1.0

0

10 N

0

9 N 1 T

10

0

5 N 5 T

0

5 N 5 T

100

9

1 T

9

1 R

Cumulative Immobilization Data and Observations in the Definitive Test

Nominal
Concentration
(% v/v Saturated Solution)

24 Hours

Cumulative Immobilized Daphnia
(Initial Population: 5 Per Replicate)

Observations

R1

R2

R3

R4

Total

%

R1

R2

R3

R4

Control

0

0

0

0

0

0

5 N

5 N

5 N

5 N

2.6

0

0

0

0

0

0

5 N

5 N

5 N

5 N

6.4

0

0

0

0

0

0

5 N

5 N

5 N

5 N

16

1

0

0

0

1

5

4 N

5 N

5 N

5 N

40

0

0

0

0

0

0

5 N

5 N

5 N

5 N

100

4

4

2

2

12

60

1 N

1 N

3 N

3 N

 

Nominal
Concentration
(% v/v Saturated Solution)

48 Hours

Cumulative Immobilized Daphnia
(Initial Population: 5 Per Replicate)

Observations

R1

R2

R3

R4

Total

%

R1

R2

R3

R4

Control

0

1

0

0

1

5

5 N

4 N

5 N

5 N

2.6

1

0

1

0

2

10

4 N

5 N

4 N

5 N

6.4

0

0

0

0

0

0

1 T 4 N

5 N

5 N

1 T 4 N

16

1

0

0

0

1

5

4 N

5 N

5 N

1 T 4 N

40

0

0

1

1

2

10

5 N

1 T 4 N

4 N

4 N

100

5

5

5

5

20

100

A/I

A/I

A/I

A/I

Water Quality Measurements

Nominal
Concentration
(% v/v Saturated Solution)

0 Hours
(Fresh Media)

24 Hours
(Old Media)

pH

mg O2/L

T°C

pH

mg O2/L

T°C

Control

R1

8.1

8.7

21

8.3

8.5

21

2.6

R1

8.1

8.8

21

8.2

8.6

21

6.4

R1

8.1

8.7

21

8.2

8.6

21

16

R1

8.1

8.8

21

8.2

8.6

21

40

R1

8.0

8.6

22

8.2

8.6

21

100

R1

8.0

8.5

22

8.2

8.6

21

 

Nominal
Concentration
(% v/v Saturated Solution)

24 Hours
(Fresh Media)

48 Hours
(Old Media)

pH

mg O2/L

T°C

pH

mg O2/L

T°C

Control

R1

8.1

8.8

21

7.9

8.8

21

2.6

R1

8.1

8.8

21

8.0

8.7

21

6.4

R1

8.0

8.7

21

8.0

8.7

21

16

R1

8.0

8.7

21

8.0

8.7

21

40

R1

8.0

8.7

21

8.0

8.7

21

100

R1

8.0

8.7

22

8.0

8.6

21

R1= Replicate 1

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Exposure of Daphnia magna to the test item has been investigated and gave the following results based on the geometric mean measured test concentrations:
The 48 h EC50 was 0.36 mg/L. 95 % confidence limits could not be determined.
The NOEC was 0.29 mg/L and the LOEC was 0.74 mg/L.