4-(1-methoxy-1-methylethyl)-1-methylcyclohexene

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:

Form 14 July 2015 to 20 August 2015

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:

according to guideline

Guideline:

other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23, December 14, 2000

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Samples of 3.0mL from the approximate centre of the test vessels were taken from all test concentrations and the control at t=0, 24 and 48 h. Samples were stored in a freezer until analysis. At the end of the exposure period, the replicates were not pooled at each concentration before sampling.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
The batch of Orange Flower Ether tested was a colourless liquid with a purity of 83.8% and not completely soluble in test medium at the concentrations tested. Test solutions were prepared in airtight closed vessels at loading rates ranging from 1.0 to 100 mg/L applying a 2-day period of magnetic stirring followed by a one-hour stabilisation period. The mixture prepared at 100 mg/L in the combined limit/range-finding test contained undissolved material and therefore, the Water Accommodated Fractions (WAFs) were collected by siphoning from all solutions. In the final test no undissolved test material was observed after the stabilisation period and therefore, no further treatment was needed. The final test solutions were all clear and colourless.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna (Straus, 1820)
- Source: In-house laboratory culture with a known history, at least third generation, obtained by acyclical parthenogenesis under specified breeding conditions. Daphnids originated 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 there was no delay in the production of the first brood.
- Age at study initiation: young daphnids with an age of < 24 hours, from parental daphnids of more than two weeks old.
- Method of breeding: start of each batch 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. Renewal of the cultures: after 7 days of cultivation half of the medium twice a week.
- Feeding during test: no

ACCLIMATION
- Acclimation period: no

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

none

Hardness:

180 mg/L (as CaCO3).

Test temperature:

20 - 21 °C

pH:

7.8 - 8.1

Dissolved oxygen:

8.1 - 9.1 mg O2/L

Nominal and measured concentrations:

- Nominal concentrations: 2.2, 4.6, 10, 22 and 46 mg/L (based on range-finding test).
- Measured concentrations (geom. mean): n.d., 1.7, 3.7, 9.9, 16 and 43 mg/L.

- Recovery: Mean measured concentrations ranged from 68 -101% of nominal concentrations (calculated by reviewer).
- Relative to initial [%] ranged from 95 - 124%.

Details on test conditions:

TEST SYSTEM
- Test vessel: 50 mL, all-glass, silanised to prevent sticking of daphnids to glass, closed airtight
- Aeration: None
- No. of organisms per vessel: 5 (one replicate vessel of the highest concentration unintentionally contained 6 daphnids, which was taken into account during calculation of effects)
- 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
- Culture medium different from test medium: Yes, Culture medium had extra trace elements, macro nutrients and vitamins added as specified in Medium M7.

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

EFFECT PARAMETERS MEASURED:
Mobility at 24 and 48 hours

PHYSICOCHEMICAL PARAMETERS MEASURED
pH and dissolved oxygen at the beginning and at the end of the test. Temperature continuously.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

15 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CI: 12 - 18 mg/L

Results with reference substance (positive control):

48-h EC50: 0.36 mg/L (with 10% effect at 0.32 mg/l and 100% effect at 0.56 mg/L). Test performed June 2015.

Reported statistics and error estimates:

The 48h-EC50-value was calculated from the weibits of the percentages of affected daphnids and the logarithms of the corresponding test substance concentrations using the maximum likelihood estimation method.

Number of introduced daphnids and incidence of immobility in the final test

Mean measured conc. (mg/L)	Incidence of immobility after:			Cumulative immobility after 48h (%)
	0h	24h	48h
0 (control)	0/20	0/20	0/20	0
1.7	0/20	0/20	0/20	0
3.7	0/20	1/20	1/20	5
9.9	0/20	2/20	8/20	40
16	0/20	0/20	8/20	40
34	0/21	18/21	21/21	100

Average exposure concentration versus nominal concentration

Loading rate (mg/L)	Measured concentrations (mg/L)			Geom. mean measured concentrations (mg/L)
	t=0h	t=24h	t=48h
0 (control)	n.d.	n.d.	n.d.	n.a.
2.2	1.62	1.69	1.68	1.7
4.6	3.62	6.62	3.74	3.7
10	9.43	10.2	10.1	9.9
22	15.8	16.3	15.0	16
46	31.3	38.8	31.2	34

Validity criteria fulfilled:

yes

Remarks:

See 'Any other information on materials and methods incl. tables)

Conclusions:

The 48h-EC50 value to Daphnia magna was 15 mg/L based on 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 under static conditions. Based on the results of a preliminary range-finding test, the following nominal concentrations were tested in the definitive test: 2.2, 4.6, 10, 22 and 46 mg/L. For each concentration and a control group, twenty Daphnia magna (less than 24 hours old) were exposed for 48 hours. One replicate vessel of the highest concentration unintentionally contained 6 instead of 5 daphnids. Therefore the highest test concentration was tested with 21 Daphnia magna. This was taken into account in the calculation of effects and therefore the test results were not affected. The exposure took place in airtight closed vessels. The incidence of immobilisation was recorded for each test and control group at 24 hours and at 48 hours. Samples taken from all concentrations at t = 0, 24 and 48 were analysed with a validated GC-FID method. Analysis of the samples taken at the start of the final test showed that some measured concentrations were <80% of the nominal concentrations. The concentrations remained stable during the 48-hour test period. Based on these results, the mean measured concentrations were calculated. The range tested based on mean measured concentrations was 1.7, 3.7, 9.9, 16 and 34 mg/L. The 48h-EC50 value to Daphnia magna was 15 mg/L based on mean measured concentrations.

Description of key information

Data on the short-term toxicity to aquatic invertebrates is available from a study according to OECD 202 and the LC50 value determined at 15 mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

15 mg/L

Additional information

The acute toxicity to aquatic invertebrates was determined in a study according to OECD TG 202 and in compliance with GLP criteria (IFF, 2015). In this study daphnids (D. magna, 20 per concentration) were exposed to nominal concentrations of 0 (control), 2.2, 4.6, 10, 22 and 46 mg/L for 48 hours under static conditions. Test concentrations were analytically verified at t=0h, t=24h and t=48h and geomean measured concentrations determined at 1.7, 3.7, 9.9, 16 and 43 mg/L. Immobilization was recorded after 24 and 48 hours exposure. A cumulative immobility of 5% was recorded at a test concentrations of 3.7 mg/L. Cumulative immobility was 40% at the 9.9 and 16 mg/L test concentrations and further increased to 100% at the highest tested concentration of 34 mg/L. Based on these findings the 48 -h EC50 value was determined at 15 mg/L based on measured concentrations.