2,4-dimethylcyclohex-3-ene-1-methyl 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:

06 January 2017 - 20 February 2017

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:

2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

2008

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

other: Guidance document on aquatic toxicity testing of difficult items and mixtures, OECD series on testing and assessment number 23

Version / remarks:

December 14, 2000

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: control and all test concentrations
- Sampling method: 6 mL were taken from the approximate centre of the test vessels at the start of the test and after 24 hours from the freshly prepared solutions; and at the first renewal (t=24h) and the end of the test (t=48 h) from the 24-hour old solutions
- Sample storage conditions before analysis: All samples were stored in a freezer (at <= -15°C) until analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: preparation of a saturated solution (direct addition) - In view of the test substance being a multi-constituent, constituted by two stereo-isomers, the test item was treated as pure and therefore a Saturated Solution was used. Preparation of test solutions started with a loading rate of 100 mg/L applying 3 days of magnetic stirring in a closed vessel at room temperature to ensure maximal dissolution of the test item in the test medium. The resulting aqueous mixture was left to stabilize for 60-89 minutes whereafter the clear and colourless Saturated Solution (SS) was siphoned off and used as the highest test concentration. Lower test concentrations were prepared by subsequent dilutions of the SS in test medium. All final test solutions were clear and colourless
- Controls: Test medium without test item or other additives
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna (Crustacea, Cladocera) (Straus, 1820)
- Strain/clone: at least third generation, obtained by a cyclical parthenogenesis under specified breeding conditions
- Source: In-house laboratory culture with a known history.
- Age of parental stock: older than two weeks (originating from a healthy stock, 2nd to 5th brood, showing no signs of stress such as mortality >20%, presence of males, ephippia or discoloured animals and without delay in the production of the first brood)
- Age of test organisms at test initiation: young daphnids < 24 hours old
- Feeding during test: no

ACCLIMATION
- Acclimation period: no

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:
Breeding: Each new batch of daphnids is started with newborn daphnids, i.e. less than 3 days old, by placing about 250 of them into 5 litres of medium (M7) in an all-glass culture vessel. The maximum age of the cultures is 4 weeks. After 7 days of cultivation half of the medium is renewed twice a week. Temperature of the medium is 18-22°C. Daphnids are fed daily a suspension of fresh water algae.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

180 mg/L (as CaCO3)

Test temperature:

19-21°C

pH:

fresh solutions: 7.8-8.0
used solutions: 7.6-8.0

Dissolved oxygen:

fresh solutions: 8.5-9.4
used solutions: 7.6-9.4

Nominal and measured concentrations:

Nominal test concentrations (defintive test): 1.0, 2.2, 4.6, 10, 22, 46 and 100% (v/v) of a saturated solution prepared at a loading rate of 100 mg/L
Corresponding mean measured concentrations: 0.83, 1.8, 3.6, 8.1, 16, 35 and 65 mg/L

Measured concentrations in freshly prepared solutions were 61-90% of nominal. Measured concentrations in used solutions were 92-102% of initial. See also field 'Any other information on results'.
Therefore effect concentrations were based on geometric mean measured concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel: 60 mL, all-glass, air-tight closed vessels with headspace reduced to minimum
- Aeration: no
- Renewal rate of test solution: yes, after 24 hours
- 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: adjusted ISO medium according to OECD 202
- Culture medium different from test medium: yes - culture medium: Elendt M7
- Intervals of water quality measurement: water temperature - continuously; pH and dissolved oxygen concentrations - at the start and the end of both renewal periods, for all concentrations and the control

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours photoperiod daily

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): mobility (at 24 and 48 hours)

RANGE-FINDING STUDY
- Test concentrations: 1.0, 10 and 100% (v/v) of a saturated solution prepared at a loading rate of 100 mg/L (measured concentration in fresh 100% saturated solutions was 75.5 mg/L (0 hours, fresh) and 89.5 mg/L (24 hours, fresh)).
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

potassium dichromate (February 2017)

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

16.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mobility

Remarks on result:

other: 95% C.I. 14.4-19.2 mg/L

Details on results:

- Immobility of control: 5% immobilization observed in the control. OECD Guideline 202 allows for 10% immobility in the control group. Consequently, responses up to 10% are not considered biologically significant.
- Other adverse effects control: no
- Effect concentrations exceeding solubility of substance in test medium: no
- Other: A dose response was observed at the three highest test concentrations. All daphnids exposed to the highest test concentration were immobilised already at 24 hours of exposure. Microscopic observations of daphnids exposed to an average concentration of 16 mg/L revealed no test item attached to their bodies after 24 hours of exposure.

Individual pH, temperature and dissolved oxygen values remained within acceptable limits throughout the duration of the study.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Dose-response test, concentrations: 0.10, 0.18, 0.32, 0.56, 1.0 and 1.8 mg/L
- 48h-EC50: 0.48 mg/L (95% C.I. 0.43 - 0.53 mg/L)

Reported statistics and error estimates:

The EC50-value was calculated at 24 and 48 hours of exposure from the weibits of the percentages of affected daphnids and the logarithms of the corresponding average exposure concentrations using the maximum likelihood estimation method.
ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used to perform the analyses.

Table 1. Incidence of immobility in the final test

Time (h)	Replicate	Average exposure concentration (mg/L)
		Control	0.83	1.8	3.6	8.1	16	35	65
0	A	5	5	5	5	5	5	5	5
	B	5	5	5	5	5	5	5	5
	C	5	5	5	5	5	5	5	5
	D	5	5	5	5	5	5	5	5
	Total introduced	20	20	20	20	20	20	20	20
24	A	1	0	0	0	0	3	2	5
	B	0	0	0	0	0	0	3	5
	C	0	0	0	0	0	0	5	5
	D	0	0	0	0	0	2	5	5
	Total immobilised	1	0	0	0	0	5	15	20
	Effect %	5	0	0	0	0	25	75	100
48	A	1	0	0	0	0	3	5	5
	B	0	0	0	0	0	2	5	5
	C	0	0	0	0	0	2	5	5
	D	0	0	0	0	0	2	5	5
	Total immobilised	1	0	0	0	0	9	20	20
	Effect %	5	0	0	0	0	45	100	100

 

No biologically relevant immobility was observed in the control and in the four lowest test concentrations throughout the test duration (OECD guideline 202 allows for 10% immobility in the control group. Consequently, responses up to 10% are not considered biologically significant).

 

Table 2. Average exposure concentration versus nominal concentration

Citrolate %SS at 100 mg/L	Measured concentration (mg/L)				Average exposure concentration (mg/L)
	t=0h (fresh)	t=24h (old)	t=24h (fresh)	t=48h (old)
1.0	0.803	0.799	0.902	0.837	0.83
2.2	1.75	1.73	1.93	1.78	1.8
4.6	3.53	3.25	3.86	3.65	3.6
10	7.78	7.83	8.49	8.13	8.1
22	16.5	15.9	17	16.4	16
46	34.4	33.4	37.6	35.9	35
100	61.3	62.4	70.2	65.7	65

 

Table 3. Final Test: measured concentrations in Test Samples

Time of sampling [hours]	Percentage of SS^1 [%]	Analyzed concentration^2 [mg/L]	Relative to initial [%]
0	0	n.d.
	1.0	0.803
	2.2	1.75
	4.6	3.53
	10	7.78
	22	16.5
	46	34.4
	100	61.3
24	0	n.d.	n.a.
(old)	1.0	0.799	100
	2.2	1.73	99
	4.6	3.25	92
	10^3	7.83	101
	22	15.9	97
	46	33.4	97
	100	62.4	102
24	0	n.d.
(fresh)	1.0	0.902
	2.2	1.93
	4.6	3.86
	10	8.49
	22	17.0
	46	37.6
	100	70.2
48	0	n.d.	n.a.
(old)	1.0	0.837	93
	2.2	1.78	92
	4.6	3.65	95
	10	8.13	96
	22	16.4	96
	46	35.9	96
	100	65.7	94

¹      Percentage of a saturated solution (SS) prepared at a loading rate of 100 mg/L.

²      Samples were stored in the freezer (≤ -15°C) until the day of analysis.

³         Sample was reinjected due to a misinjection^.

n.d.Not detected.

n.a.Not applicable.

 

Measured concentrations in the 100% saturated solution (prepared at a loading rate of 100 mg/L) were 61% and 70% of nominal.

Validity criteria fulfilled:

yes

Remarks:

see 'Overall remarks'

Conclusions:

The 48h-EC50 value to Daphnia magna was 16.6 mg/L based on geometric mean measured concentrations.

Executive summary:

A study was performed to assess the acute toxicity of the substance to Daphnia magna. The study was conducted in accordance with OECD Guideline for Testing of Chemicals No. 202 and GLP under semi-static conditions. Based on the results of a range-finding test, the following nominal concentrations were tested in the definitive test: untreated control, 1.0, 2.2, 4.6, 10, 22 and 46% (v/v) dilutions of a saturated solution prepared at loading rate of 100 mg/L, as well as the 100% saturated solution. For each concentration and a control group, twenty Daphnia magna (less than 24 hours old) were exposed for 48 hours in air-tight closed vessels. The incidence of immobilisation was recorded for each treatment group and controls at 24 hours and at 48 hours. Samples taken from all concentrations at the start and the end of both 24 -hour renewal periods were analysed with a validated GC-FID method. Initial measured concentrations in the freshly prepared 100% saturated solution were 61% and 70% of the nominal loading rate. Measured concentrations in used test solutions ranged from 92 -102% of initial in the first renewal period, and from 92 -96% of initial in the second renewal period. Since the test concentrations in the freshly prepared 100% saturated solutions were not within 80 -120% of nominal, geometric mean measured concentrations were calculated. The range tested based on geometric mean measured concentrations was 0.83, 1.8, 3.6, 8.1, 16, 35 and 65 mg/L. The 48h-EC50 value to Daphnia magna was 16.6 mg/L based on geometric mean measured concentrations.

Description of key information

A study was performed to assess the acute toxicity of the substance to Daphnia magna. The study was conducted in accordance with OECD Guideline for Testing of Chemicals No. 202 and GLP under semi-static conditions. Based on the results of a range-finding test, the following nominal concentrations were tested in the definitive test: untreated control, 1.0, 2.2, 4.6, 10, 22 and 46% (v/v) dilutions of a saturated solution prepared at loading rate of 100 mg/L, as well as the 100% saturated solution. For each concentration and a control group, twenty Daphnia magna (less than 24 hours old) were exposed for 48 hours in air-tight closed vessels. The incidence of immobilisation was recorded for each treatment group and controls at 24 hours and at 48 hours. Samples taken from all concentrations at the start and the end of both 24 -hour renewal periods were analysed with a validated GC-FID method. Initial measured concentrations in the freshly prepared 100% saturated solution were 61% and 70% of the nominal loading rate. Measured concentrations in used test solutions ranged from 92 -102% of initial in the first renewal period, and from 92 -96% of initial in the second renewal period. Since the test concentrations in the freshly prepared 100% saturated solutions were not within 80 -120% of nominal, geometric mean measured concentrations were calculated. The range tested based on geometric mean measured concentrations was 0.83, 1.8, 3.6, 8.1, 16, 35 and 65 mg/L. The 48h-EC50 value to Daphnia magna was 16.6 mg/L based on geometric mean measured concentrations.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

16.6 mg/L