Registration Dossier

Administrative data

Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 01 November 2015 and 11 November 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 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)

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:
Water samples were taken from the control and 32% v/v saturated solution test vessel at 0 and 72 hours from fresh media and at 24 and 96 hours from old media for quantitative analysis. The samples were stored frozen prior to analysis.
Duplicate samples and samples at 24 (fresh media), 48 (old and fresh media) and 72 hours (old media) were taken and stored frozen for further analysis if necessary.

Test solutions

Vehicle:
no
Details on test solutions:
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 filter (first approximate 2 liters discarded in order to pre-condition the filter) to give a 100% v/v saturated solution. A dilution was prepared from the 100% v/v saturated solution to produce the required test concentration of 32% v/v saturated solution.

The concentration and stability of the test item in the test preparations were verified by chemical analysis of the fresh media at 0 and 72 hours and of the old media at 24 and 96 hours

Test organisms

Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
The test was carried out using juvenile rainbow trout. Fish were obtained from Brow Well Fisheries Limited, Hebden, near Skipton, Yorkshire, UK and maintained in house since
07 October 2015. Fish were maintained in a glass fiber tank with a "single pass" water renewal system. Fish were acclimatized to test conditions from 26 October 2015 to 2 November 2015. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods.

The water temperature was controlled at 12 to 13 °C with a dissolved oxygen content of greater than or equal to 9.2 mg O2/L. These parameters were recorded daily. The stock fish were fed commercial trout pellets which was discontinued approximately 24 hours prior to the start of the definitive test. There was no mortality in the 7 days prior to the start of the test and the fish had a mean standard length of 4.1 cm (sd = 0.4) and a mean weight of 0.97 g (sd = 0.26) at the end of the definitive test. Based on the mean weight value this gave a loading rate of 0.34 g bodyweight/liter (static volume).

The diet and diluent water are considered not to contain any contaminant that would affect the integrity and outcome of the study.

Study design

Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h

Test conditions

Test temperature:
The water temperature was recorded daily throughout the test. The measurements at 0 hours, and after each test media renewal at 24, 48 and 72 hours, represent those of the freshly prepared test preparations while the measurements taken prior to each test media renewal, and on termination of the test after 96 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 13 to 14 °C throughout the test
pH:
The pH was recorded daily throughout the test. The measurements at 0 hours, and after each test media renewal at 24, 48 and 72 hours, represent those of the freshly prepared test preparations while the measurements taken prior to each test media renewal, and on termination of the test after 96 hours, represent those of the used or 24-Hour old test preparations. The pH was measured using a Hach Flexi handheld meter. There were no treatment related differences for pH
Dissolved oxygen:
Thedissolved oxygen concentrations were recorded daily throughout the test. The measurements at 0 hours, and after each test media renewal at 24, 48 and 72 hours, represent those of the freshly prepared test preparations while the measurements taken prior to each test media renewal, and on termination of the test after 96 hours, represent those of the used or 24-Hour old test preparations. The dissolved oxygen concentrations were measured using a Hach Flexi handheld meter. There were no treatment related differences for oxygen concentration.
Nominal and measured concentrations:
0.18 mg/L (equivalent to a nominal concentration of 32% v/v saturated solution)
Details on test conditions:
Test water
The test water used for the definitive test was the same as that used to maintain the stock fish.

Laboratory tap water was dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 1248D Duplex Water Softener) giving water with a total hardness of approximately 140 mg/L as CaCO3. After dechlorination and softening the water was passed through a series of computer controlled plate heat exchangers to achieve the required temperature.

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.


Definitive Test
In accordance with the recommendations of REACh, the test was conducted according to the threshold approach recommended by ECHA. Using this approach the lowest EC50 value from either the Algal Growth Inhibition study or Acute Toxicity to Daphnia magna study is set as the threshold concentration and a “Limit test” is conducted at this threshold concentration. If no mortalities are observed this indicates that fish are not the most sensitive species and that the LC50 is greater than the threshold concentration. The EC50 value obtained from the Algal Growth Inhibition study (Study Number 41402864) was the lowest of these two EC50 values and hence the test was conducted at a single concentration of 0.18 mg/L (equivalent to a nominal concentration of 32% 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 filter (first approximate 2 liters discarded in order to pre-condition the filter) to give a 100% v/v saturated solution. A dilution was prepared from the 100% v/v saturated solution to produce the required test concentration of 32% v/v saturated solution.

The concentration and stability of the test item in the test preparations were verified by chemical analysis of the fresh media at 0 and 72 hours and of the old media at 24 and 96 hours.


Exposure Conditions
In the definitive test, 25-30 liter glass exposure vessels containing 20 liters of test media were used for each control and test concentration. At the start of the test seven fish were placed in each test vessel at random, in the test preparations. The test vessels were then covered to reduce evaporation and maintained at approximately 14 °C in a temperature controlled room with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for a period of 96 hours. The test vessels were aerated via narrow bore glass tubes.

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

A semi-static test regime was employed in the test involving a daily renewal of the test preparations to prevent the build up of nitrogenous waste products.


Evaluations
Test Organism Observations
Any mortalities and sub-lethal effects of exposure were recorded at 3, 6, 24, 48, 72 and 96 hours after the start of exposure. The criteria of death were taken to be the absence of both respiratory movement and response to physical stimulation.
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 0.22 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Key result
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
0.22 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Details on results:
Mortality Data
There were no mortalities in 7 fish exposed to a geometric mean measured test concentration of 0.22 mg/L for a period of 96 hours.
The No Observed Effect Concentration (NOEC) was 0.22 mg/L.

Sub-Lethal Effects
There were no sub-lethal effects of exposure observed in 7 fish exposed to a geometric mean measured test concentration of 0.22 mg/L for a period of 96 hours.
Reported statistics and error estimates:
Statistical Analysis
An estimate of the LC50 values was given by inspection of the mortality data.

Geometric Mean Measured Test Concentrations
A geometric mean measured concentration was calculated for the 0 to 24 and 72 to 96 Hour exposure periods as follows using the measured test concentrations:

GM = √(C0 – C1)

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

The average of the geometric mean measured concentrations was then calculated as follows:

GMav = GM1GM2/2

Where:
GMav = average geometric mean measured test concentration (mg/L)
GM1 = geometric mean measured concentration for the first exposure period (mg/L)
GM2 = geometric mean measured concentration for the second exposure period (mg/L)

Any other information on results incl. tables

Sublethal observations / clinical signs:

Verification of Test Concentrations

Analysis of the fresh test preparations at 0 and 72 hours showed measured test concentrations of 0.39 and 0.17 mg/L respectively. Analysis of the old test media at 24 and 96 hours showed measured test concentrations of 0.18 and 0.17 mg/L respectively. Given these results, it was considered appropriate to calculate the results based on the geometric mean measured test concentration only in order to give a “worst case” analysis of the data.

 

The geometric mean measured test concentration was determined to be:

 

Nominal Test Concentration
(% v/v Saturated Solution)

Geometric Mean Measured Test Concentration (mg/L)

0 – 24 Hour

72 – 96 Hour

Average

32

0.26

0.17

0.22

 

Mortality Data

There were no mortalities in 7 fish exposed to a geometric mean measured test concentration of 0.22 mg/L for a period of 96 hours. Inspection of the mortality data gave the following results:

 

Time (h)

LC50(mg/L)

3

>0.22

6

>0.22

24

>0.22

48

>0.22

72

>0.22

96

>0.22

Validation Criteria

The test was considered to be valid given that none of the control fish died or showed signs of stress during the test and that the oxygen concentration at the end of the test was greater than or equal to 60% of ASV (6.1 mg O2/L) in the control and test vessels.

Observations on Test Item Solubility

The test item preparations were observed to be clear colorless solutions throughout the test.

Cumulative Mortality Data in the DefinitiveTest

Nominal
Concentration
(% v/v Saturated Solution)

Geometric Mean Measured Test
Concentration
(mg/L)

Cumulative Mortality (Initial Population = 7)

%

Mortality

3
Hours

6
Hours

24 Hours

48 Hours

72 Hours

96 Hours

96
Hours

Control

0

0

0

0

0

0

0

32

0.22

0

0

0

0

0

0

0

Water Quality Measurements

Nominal Concentration
(% v/v Saturated Solution)

Time (Hours)

0 Hours (Fresh Media)

24 Hours (Old Media)

pH

mg O2/L

T ºC

pH

mg O2/L

T°C

Control

7.3

10.2

13

7.8

9.7

14

32

7.5

10.2

14

7.9

9.7

14

 

Nominal Concentration
(% v/v Saturated Solution)

Time (Hours)

24 Hours (Fresh Media)

48 Hours (Old Media)

pH

mg O2/L

T ºC

pH

mg O2/L

T°C

Control

7.6

9.7

13

7.9

10.0

14

32

7.7

9.8

14

7.9

9.7

14

 

Nominal Concentration
(% v/v Saturated Solution)

Time (Hours)

48 Hours (Fresh Media)

72 Hours (Old Media)

pH

mg O2/L

T ºC

pH

mg O2/L

T°C

Control

7.6

9.7

14

7.9

9.8

14

32

7.7

9.6

14

7.9

9.8

14

 

Nominal Concentration
(% v/v Saturated Solution)

Time (Hours)

72 Hours (Fresh Media)

96 Hours (Old Media)

pH

mg O2/L

T ºC

pH

mg O2/L

T°C

Control

7.6

9.7

13

7.8

9.8

14

32

7.6

9.7

 

14

7.9

9.8

14

 

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The acute toxicity of the test item to the freshwater fish rainbow trout has been investigated and based on the geometric mean measured test concentration gave a 96-Hour LC50 of greater than 0.22 mg/L. The No Observed Effect Concentration was 0.22 mg/L.