2,6-dimethyloct-7-en-2-yl formate

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:

22 February 2018 - 23 July 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

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Remarks:

HPLC - UV method.

Details on sampling:

- Concentrations: 26.0, 36.4, 51.0, 71.3 and 100.0% w/v saturated test item solutions.
- Sampling method: Samples were taken from the test solutions at the applied concentration levels and from the control solution at 0, 24 and 48h. 310 mL of test sample from additional replicate of each group was drawn and divided into equal portion (155 mL in each portion) at 0, 24 and 48 h. One portion was sent for test concentration analysis and the second portion was stored at -20 ± 5 ºC temperature till the study completion. Active ingredient concentration in water was determined using a validated analytical method.
- Sample storage conditions before analysis: not required.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 8000 mL of 100% w/v saturated concentration prepared by mixing 100 mg of test item with 1L of reconstituted water individually in 1 L of 8 glass bottle (Stock solution A, B, C, D, E, F, G, and H). After mixing the test item, it was stirred on magnetic stirrer for 180 min, transferred to separating funnel having capacity of 1L and allowed to equilibrate for 30 min. After 30 min, 100% saturated solution collected and used for study. Volumes of 80.6, 112.84, 158.1, 221.0 and 310.0 mL from 100% w/v saturated concentration prepared at 100 mg dihydromyrcenyl formate/L and diphnids with 0.5 mL reconstituted water and diluted to 310 mL with reconstituted water in screw capped 250 mL glass bottles to obtain the nominal test concentrations of 26.0, 36.4, 51.0, 71.3 and 100.0% w/v saturated concentration prepared at 100 mg dihydromyrcenyl formate/L.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Strain: Daphnia magna
- Source: MicroBio Test Inc, Kleimoer 15, 9030 Mariakerke (Gent), Belgium
- Age of the animals: less than 24 h old at the beginning of the test.
- Feeding during test: No

ACCLIMATION
- Acclimation period: 48h
- Acclimation conditions (same as test or not): yes
- Type and amount of food: live algal cells (Pseudokirchneriella subcapitata)

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

179.2 mg/L (as CaCO3)

Test temperature:

18.6 – 20.3ºC

pH:

6.36 – 7.39

Dissolved oxygen:

8.04 – 8.41 mg/L.

Nominal and measured concentrations:

Nominal concentrations: 0 (control), 26.0, 36.4, 51.0, 71.3 and 100.0% w/v saturated test item solutions.
Measured concentrations: 0, 12.76, 18.50, 22.22, 33.84 and 46.95 mg/L (geometric mean, measured).

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL screw capped glass bottles
- Material, size, headspace, fill volume: glass, 250 mL (metric capacity), fill volume 310 mL.
- Aeration: No
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water (ISO medium, according to OECD 202)

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16-hour light and 8-hour dark cycle
- Light intensity: 1300 – 1320 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
The immobility or mortality of the Daphnia was determined by visual observation 24 and 48 hours after the start of the test. Those animals not able to swim within 15 seconds after gentle agitation of the test beaker are considered to be immobile.

RANGE-FINDING STUDY
- Test concentrations: Non GLP research trial study was conducted with test concentrations of 0.0 (control), 0.1, 1.0, 10.0, 25.0, 50.0 and 100.0% w/v saturated concentration prepared at 100 mg/L.
- Results used to determine the conditions for the definitive study: The percent immobility observed were 0, 0, 0, 0, 0, 20 and 100% at the test concentrations of 0.0 (control), 0.1, 1.0, 10.0, 25.0, 50.0 and 100.0% w/v saturated concentration prepared at 100 mg/L, respectively. Based on the results of non-GLP research study, the test concentrations selected for the main study were 26.0, 36.4, 51.0, 71.3 and 100.0% w/v saturated concentration prepared at 100 mg dihydromyrcenyl formate/L (geometric factor 1.4).

Reference substance (positive control):

yes

Remarks:

(Potassium dichromate, last test 01/02/2018, ref: 502-3-07-19120)

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

29.93 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

12.76 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

18.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Behavioural abnormalities: No abnormal behaviour, appearance or any other effect on test animals were detected. At 24 and 48h, lethargy was observed in G3 (36.4% w/v saturated concentration prepared at 100 mg dihydromyrcenyl formate/L), G4 (51.0% w/v saturated concentration prepared at 100 mg dihydromyrcenyl formate/L) and G5 (71.3% w/v saturated concentration prepared at 100 mg dihydromyrcenyl formate/L. 100% immobility was observed in G6 (100% w/v saturated concentration prepared at 100 mg dihydromyrcenyl formate/L). No immobility and behavioural symptoms was observed in G2 (26% w/v saturated concentration prepared at 100 mg dihydromyrcenyl formate/L) and G1 (control) groups. No sign of disease or stress, e.g., discolouration or unusual behaviour such as trapping at the surface of water was observed in any replicate of the control group.
- Mortality of control: 0/20

Results with reference substance (positive control):

- Results with reference substance valid? Yes.
Potassium dichromate is tested to demonstrate satisfactory test conditions; last test 01/02/2018, Study No.: 502-3-07-19120.

Table 1. Immobility Data

Group	Nominal Concentration (% w/v saturated concentration prepared at 100 mg/L)	Geometric mean measured concentration (mg a.i./L)	N° of Replicates	N° of Daphnids/ group	Immobility N° and % at
					0 h		24 h		48 h
					N°	%	N°	%	N°	%
G1	0.0 (Control)	-	4	20	0	0	0	0	0	0
G2	26.0	12.76	4	20	0	0	0	0	0	0
G3	36.4	18.50	4	20	0	0	0	0	1	5
G4	51.0	22.22	4	20	0	0	0	0	6	30
G5	71.3	33.84	4	20	0	0	0	0	12	60
G6	100.0	46.95	4	20	0	0	20	100	20	100

Key: h = Hour, - = Not Applicable

 

Table 2. Mean Values of Physico-Chemical Parameters of Test Media

Groups	Nominal Concentration (% w/v saturated concentration prepared at 100 mg/L)	pH				Temperature (°C)				Dissolved Oxygen (%)				Total Hardness (as CaCO3) mg/L water
		Initial		Final		Initial		Final		Initial		Final
		Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD	179.2
G1	0.0 (Control)	7.05	0.039	6.90	0.178	20.0	0.13	18.9	0.05	8.33	0.057	8.25	0.053
G2	26.0	7.16	0.017	7.06	0.123	20.0	0.26	18.8	0.17	8.28	0.100	8.16	0.110
G3	36.4	7.21	0.013	7.10	0.084	20.1	0.13	18.8	0.10	8.28	0.050	8.20	0.038
G4	51.0	7.25	0.008	7.19	0.166	19.9	0.13	18.8	0.10	8.28	0.054	8.22	0.048
G5	71.3	7.36	0.018	7.17	0.114	20.0	0.21	18.7	0.15	8.28	0.073	8.21	0.108
G6	100.0	7.37	0.021	6.85	0.363	20.0	0.10	18.9	0.06	8.27	0.087	8.15	0.088

Key: SD = Standard Deviation

 

Table 3. Geometric Mean of Dihydromyrcenyl Formate

Groups	Nominal Concentration (% w/v saturated concentration prepared at 100 mg/L)	Measured Concentration ofDihydromyrcenyl Formate (mg a.i./L)			Geometric Mean (mg a.i./L) 24 h	Geometric Mean (mg a.i./L) 48 h
		0 h	24 h	48 h
G1	0.0 (Control)	-	-	-	-	-
G2	26.0	14.3526300	12.9876800	11.1409000	13.65	12.76
G3	36.4	20.7413300	18.5342600	16.4639100	19.61	18.50
G4	51.0	24.2240400	21.9960300	20.5892300	23.08	22.22
G5	71.3	36.9503500	33.8285700	31.0012300	35.36	33.84
G6	100.0	49.9790900	45.4520300	45.5502300	47.66	46.95

Key:     h = Hour, GM = Geometric mean

               Formula for Calculating Geometric Mean Exposure Concentration

 

Table 4. Relationship between Percent Immobility and Concentration ofDihydromyrcenyl Formate

Exposure Time (h)	EC50Value (% w/v saturated concentration prepared at 100 mg/L)	95% Confidence Interval (mg/L)		Regression Equation (y = a + bx)
		Lower Limit	Upper Limit
48	63.9	54.7	76.4	y = -6.7 + 6.5x

 

Calculation based on Geometric Mean Concentration

Exposure Time (h)	EC50Value (mg a.i./L)	95% Confidence Interval (mg a.i./L)		Regression Equation (y = a + bx)
		Lower Limit	Upper Limit
48	29.93	25.34	35.35	y = -4.92 + 6.72x

Key: h = Hour, y = Probit of immobility, x = Log concentration of Dihydromyrcenyl Formate, a = Intercept, b = Slope

 

Validity criteria fulfilled:

yes

Remarks:

There was no immobilization in the control group and the dissolved oxygen concentration at the end of the test in control and test vessels was ≥ 8.04 mg/L.

Conclusions:

The test item has a 48h-EC50 = 29.93 mg/L (measured, geom. mean), a NOEC = 12.76 mg/L, and a LOEC = 18.50 mg/L, based on mobility.

Executive summary:

An acute immobilisation test on Daphnia magna was performed in accordance with OECD Guideline 202, following GLP. Twenty animals, divided into four groups of 5 daphnids each, were exposed to the test item or the control for 48 h. Based on the results obtained in a non-GLP preliminary range-finding test, the animals were exposed to 0 (control), 26.0, 36.4, 51.0, 71.3 and 100.0% w/v saturated test item solutions in a static system, since the test item was not soluble in water. As the test item was found to be volatile, all tests were performed in 250 mL (metric capacity) screw-capped glass bottles with 310 mL of medium to reduce the headspace and thus avoid substance loss from the test media. Test concentrations were analytically determined at every 24h interval by a validated HPLC-UV method. As the deviation from the initial values was greater than 20% from the nominal, measured concentrations are reported: 0, 12.76, 18.50, 22.22, 33.84 and 46.95 mg/L (geometric mean). A positive control (potassium dichromate) was performed to ensure the quality of the results. All validity criteria were met. The test item has a 48h-EC50 = 29.93 mg/L (measured, geom. mean), a NOEC = 12.76 mg/L, and a LOEC = 18.50 mg/L, based on mobility.

Description of key information

Key study: Method according to OECD 202. GLP study. The 48h-EC50 of the test item in Daphnia magna was found to be 29.93 mg/L (measured, geom. mean), the NOEC = 12.76 mg/L, and the LOEC = 18.50 mg/L, based on mobility.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

29.93 mg/L

Additional information

Key study. Acute immobilisation test on Daphnia magna according to OECD 202, GLP study. Based on the results obtained in a preliminary range-finding test, 4 groups of 5 daphnids each were exposed to 0 (control), 26.0, 36.4, 51.0, 71.3 and 100.0% w/v saturated test item solutions in a static system for 48 h. As the test item was found to be volatile, all tests were performed in screw-capped glass bottles and reduced headspace to avoid substance loss from the test media. Test concentrations were analytically determined at 24h intervals by a validated HPLC-UV method. As the deviation from the initial values was greater than 20% from the nominal, results are based on measured concentrations (geometric mean). A positive control (potassium dichromate) was performed to ensure the quality of the results. All validity criteria were met. The test item has a 48h-EC50 = 29.93 mg/L (measured, geom. mean), a NOEC = 12.76 mg/L, and a LOEC = 18.50 mg/L, based on mobility.