Myrcenyl acetate

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 August 2016 - 27 August 2016

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)

Version / remarks:

April 2004

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

No. 440/2008

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Verification of Test Concentrations:
Samples were taken from the control and each test group from the freshly prepared bulk test preparation at 0 and 24 hours and from the pooled replicates of the old or expired test media at 24 and 48 hours for quantitative analysis.
Duplicate samples were taken and stored frozen for further analysis if necessary.

Storage:
The samples were analyzed on the day of receipt.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
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.
The results obtained from the preliminary media preparation trial conducted indicated that a dissolved test item concentration of approximately 41 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.

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 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 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:
Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 6.25, 12.5, 25, 50 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 filter (first approximate 1 liter 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 6.25, 12.5, 25 and 50% v/v saturated solution.

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

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
The test was carried out using 1st instar Daphnia magna derived from in-house laboratory cultures.
- Feeding during test: Daphnids received no food during exposure and the test vessels were not aerated.


METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:
Adult daphnia were maintained in 150 mL glass beakers containing 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:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

none

Hardness:

The reconstituted water had an approximate theoretical total hardness of 250 mg/L as CaCO3.

Test temperature:

21 - 22 °C

pH:

7.6 - 8.1

Dissolved oxygen:

8.5 - 9.2 mg O2/L

Nominal and measured concentrations:

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.
Chemical analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.042 to 43 mg/L, whilst analysis of the old or expired test preparations at 24 hours showed that measured concentrations of between 0.033 and 36 mg/L were obtained.

No significant 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.

Definitive test:
Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 6.25, 12.5, 25, 50 and 100% v/v saturated solution.

Analysis of the freshly prepared test preparations at 0 and 24 hours showed measured test concentrations to range from 2.2 to 43 mg/L. There was no significant change in the measured concentrations of the old or expired media at 24 and 48 hours and so the results are based on mean measured concentrations of the freshly prepared media.

The mean measured concentrations of freshly prepared media are presented under "Any other information on results incl. tables".

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL ground glass stoppered conical flasks
- Type: closed
- Fill volume: approximately 100 mL, test vessels were completely filled and sealed
- Aeration: No
- Type of flow-through: No
- Renewal of test solution: Semi-static conditions: 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.
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
Reconstituted water - ISO medium
Ingredient: Final Concentration (mg/L)
CaCl2.2H2O 294
MgSO4.7H2O 123
NaHCO3 65
KCl 5.8
The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl and was aerated until the dissolved oxygen concentration was approximately air-saturation value.
The reconstituted water had an approximate theoretical total hardness of 250 mg/L as CaCO3.


- Culture medium different from test medium: Adult daphnia were maintained in 150 mL glass beakers containing Elendt M7 medium.

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods.
- Light intensity: between 200 and 1200 Lux

EFFECT PARAMETERS MEASURED:
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.

Water quality: The water temperature, pH and 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 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.

RANGE-FINDING TEST:
Cummulative immobilization data are presented under "Any other information on materials and methods".

Reference substance (positive control):

yes

Remarks:

potassium dichromate (Conducted between 29 February 2016 and 02 March 2016)

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

6.3 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other:

Remarks:

95% CI 4.9 - 8.0 mg/L

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

11 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other:

Remarks:

95% C.L.: 8.9 - 13 mg/L

Details on results:

Cumulative immobilization data and other observations from the exposure of Daphnia magna to the test item and control solutions during the definitive test are given under "Any other information on results incl. tables".

The No Observed Effect Concentrations after 24 and 48 hours exposure were 4.8 and 2.4 mg/L respectively. The Lowest Observed Effect Concentrations after 24 and 48 hours exposure were 9.8 and 4.8 mg/L respectively

- Sub-Lethal effects:
Sub-lethal effects of exposure were observed in the 12.5 and 25% v/v saturated solution test groups. These responses were reduced movement (see Any other information on results incl. tables).

- Water Quality Criteria:
Temperature was maintained at 21 oC to 22 oC throughout the test, while there were no treatment related differences for oxygen concentration or pH.
The pH was within the range of 7.6 - 8.1 and the oxygen concentrations was within the range of 8.5 - 9.2 mg O2/L.
Throughout the test the light intensity was observed to be in the range 543 to 607 Lux.

- Observations on Test Item Solubility:
At the start and throughout the test all control and test solutions were observed to be clear colorless solutions.

Results with reference substance (positive control):

A 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.

The results from the positive control with potassium dichromate were within the normal range for this reference item.

Exposure conditions for the positive control were similar to those in the definitive test.
Analysis of the immobilization data by the 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 results presented under "Any other information on results incl. tables"

Reported statistics and error estimates:

The slopes and their standard errors of the response curves at 24 and 48 hours were 6.3 (SE = 0.04) and 3.4 (SE = 0.05) respectively.

The mean measured concentrations of the freshly prepared media in the definitive test were determined to be:

Nominal Test Concentration (% v/v Saturated Solution)	Mean Measured Concentration of the Freshly Prepared Media (mg/L)
6.25	2.39
12.5	4.81
25	9.83
50	20.1
100	42.2

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	1	0	1	5	5 N	5 N	4 N	5 N
6.25	0	0	0	0	0	0	5 N	5 N	5 N	5 N
12.5	0	1	0	0	1	5	5 N	4 N	5 N	5 N
25	1	0	2	3	6	30	4 R	5 R	3 R	2 R
50	5	5	5	5	20	100	A/I	A/I	A/I	A/I
100	5	5	5	5	20	100	A/I	A/I	A/I	A/I

 

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	0	1	0	1	5	5 N	5 N	4 N	5 N
6.25	0	0	1	1	2	10	5 N	5 N	4 N	4 N
12.5	3	1	2	1	7	35	2 R	4 R	3 R	4 R
25	4	4	2	3	13	65	1 R	1 R	3 R	2 R
50	5	5	5	5	20	100	A/I	A/I	A/I	A/I
100	5	5	5	5	20	100	A/I	A/I	A/I	A/I

R₁– R₄= Replicates 1 to 4

N = No sub-lethal effects observed

R = Reduced movement

A/I = All daphnia immobilized

Positive control:

Time Point (Hours)	EC50 (mg/L)	95% Confidence Limits (mg/L)			No Observed Effect Concentration (NOEC) (mg/L)	Lowest Observed Effect Concentration (LOEC) (mg/L)
24	0.83	0.70	-	0.98	0.56	1.0
48	0.64	Not possible to determine			0.56	1.0

Validity criteria fulfilled:

yes

Remarks:

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.

Conclusions:

The 48h-EC50 value to Daphnia magna was 6.3 mg/L based on mean measured concentrations of the freshly prepared media.

Executive summary:

A study was performed to assess the acute toxicity of the substance to Daphnia magna. The study was conducted in compliance with OECD TG 202 and GLP. Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test item at nominal concentrations of 6.25, 12.5, 25, 50 and 100% v/v saturated solution for 48 hours under semi-static test conditions. Immobilization and any adverse reactions to exposure were recorded after 24 and 48 hours. Chemical analysis of the freshly prepared test preparations at 0 and 24 hours showed measured test concentrations to range from 2.2 to 43 mg/L. There was no significant change in the measured concentrations in the old or expired media at 24 and 48 hours and so the results are based on mean measured concentrations of the freshly prepared media. The range tested based on mean measured concentrations of the freshly prepared media was 2.39, 4.81, 9.83, 20.1, 42.2 mg/L. The 48-hour EC50 value to Daphnia magna was 6.3 mg/L based on the mean measured concentrations of the freshly prepared media.

Description of key information

A study was performed to assess the acute toxicity of the substance to Daphnia magna. The study was conducted in compliance with OECD TG 202 and GLP. Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test item at nominal concentrations of 6.25, 12.5, 25, 50 and 100% v/v saturated solution for 48 hours under semi-static test conditions. Immobilization and any adverse reactions to exposure were recorded after 24 and 48 hours. Chemical analysis of the freshly prepared test preparations at 0 and 24 hours showed measured test concentrations to range from 2.2 to 43 mg/L. There was no significant change in the measured concentrations in the old or expired media at 24 and 48 hours and so the results are based on mean measured concentrations of the freshly prepared media. The range tested based on mean measured concentrations of the freshly prepared media was 2.39, 4.81, 9.83, 20.1, 42.2 mg/L. The 48-hour EC50 value to Daphnia magna was 6.3 mg/L based on the mean measured concentrations of the freshly prepared media.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

6.3 mg/L