1-cyclohexylethyl (E)-but-2-enoate

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:

27 Jul - 11 Oct 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:

2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Federal Office for the Environment (FOEN)

Analytical monitoring:

yes

Details on sampling:

- Sampling method: duplicate samples were taken from the control and the dilutions 1:2.2, 1:46, 1:10 and 1:22 from test start and end (48 h) of both test medium renewal periods. The last sample was taken after 24 h from the highest test concentration (dilution 1:2.2), since all daphnids were dead at that observation time at this concentration. The samples of the lowest test concentration (dilution 1:46) were not analyzed since these concentration was below the 48-hour NOEC.
- Sample storage conditions before analysis: Immediately after sampling, acetonitrile (10 mL acetonitrile per 10 mL sample volume) was added to each sample to stabilize the latter during the storage period. Thereafter, all samples were deep-frozen (at about -20 °C).

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: As the test item is a volatile substance, the test was performed using glass tubes completely filled with test medium that were tightly sealed with glass stoppers to avoid losses of test item by evaporation. The test item was mixed in test water at a loading rate of 100 mg/L, and was intensively stirred for 3 hours in a closed vessel. After stirring the emulsion was filtered through a 0.45 μm membrane filter.
- Eluate: no
- Differential loading: The dilutions 1:2.2, 1:4.6, 1:10, 1:22 and 1:46 of the undiluted filtrate were used as test media.
- Controls: yes (test medium control)
- Evidence of undissolved material: It was assumed (and visually checked by the Tyndall effect) that the lower phase of the water column contained dissolved test item only and therefore no additional filtration was applied.
- Others:
At the start of the test (Day 0) and at the test medium renewal (Day 1) an emulsion with a loading rate of 100 mg/L was prepared by weighing 232 and 235 mg of the test item in 2320 and 2350 mL test water, respectively. The test item was mixed into test water by intensive stirring for three hours in the dark in a closed stirring vessel to dissolve a maximum of test item in test water.After stirring, the emulsion was allowed to separate for 30 minutes and thereafter the lower phase of the water column still containing a little amount of undissolved test item was filtered through a 0.45 μm membrane filter (Whatman, NC45). As a pre-caution, the filter was preconditioned with 500 mL filtrate to avoid losses of dissolved test item due to adsorption on the filter material. The undiluted filtrate was used as a stock solution for preparation of the highest test medium concentration dilution 1:2.2). It was further diluted with test water to prepare the test media with the lower test concentrations.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: water flea
- Strain/clone: Straus
- Feeding during test: none
- Food type: the green algae Desmodesmus subspicatus CHODAT, Strain No. 86.81 SAG or a mixture of this algal suspension and a commercial fish diet (Tetra Min®Hauptfutter, supplied by TETRA-Werke, 49324 Melle / Germany)
- Others: At the start of the test, the organisms used in the test were 6-24 hours old and were not first brood progeny.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

250 as CaCO3 mg/L

Test temperature:

20 and 21 °C

pH:

control: 7.8 - 7.9
test item concentrations 7.7 - 7.9

Dissolved oxygen:

control 8.4 - 8.7
test item concentrations 8.3 - 8.7

Nominal and measured concentrations:

nominal: dilutions 1:2.2, 1:4.6, 1:10, 1:22, 1:46 of the undiluted filtrate (test item emulsion with a loading rate of 100 mg/L)
mean measured: 8.9, 4.4, 2.0, 0.87 mg/L, n.a.

Details on test conditions:

TEST SYSTEM
Test vessel
- Type: closed
- Material, size, headspace, fill volume: glass tubes was filled completly (without headspace) with 60 mL test medium and tightly sealed with glass stoppers to avoid losses of the volatile substance by evaporation (closed system).
- Aeration: no aeration
- Renewal rate of test solution: after 24 h
- 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: test water according to ISO Standard 6341 (1996)
- Alkalinity: 0.8 mmol/L
- Culture medium different from test medium: no data
- Intervals of water quality measurement: start and end of the test medium renewal periods, the pH values, dissolved oxygen concentrations and water temperature were determined in each treatment. At the same observation dates, the appearance of the test media was visually controlled and documented at the same time.

OTHER TEST CONDITIONS
- Photoperiod: 16-hour light/8-hour dark cycle and a 30-minute transition period
- Light intensity: between 15 and 18 μmol m-2 s-1

EFFECT PARAMETERS MEASURED:
- Immobilization after 24 and 48 h

RANGE-FINDING STUDY
- Test concentrations: control, dilution 1:5 and 1:20 of the undiluted filtrate (test item emulsion with a loading rate of 100 mg/L) and the undiluted filtrate itself were tested
- Results used to determine the conditions for the definitive study: 0% immobilization in the control, dilution 1:5 and 1:20 of the undiluted filtrate. 100% immobilization with the undiluted filtrate (with a loading rate of 100 mg/L)

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

2.1 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95%-CL:

Remarks:

1.8 - 2.9 mg/L

Duration:

48 h

Dose descriptor:

EC0

Remarks:

and NOEC

Effect conc.:

0.87 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

4.4 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Other biological observations: No daphnids showed immobilization or other signs of disease or stress.
- Mortality of control: 0
- Any observations that might cause a difference between measured and nominal values: No remarkable observations were made concerning the appearance of the test media. All test media were clear solutions throughout the test medium renewal periods.
- Other: Biological and analytical results are summarized in the tables 1-2 within the section "Any other information on results incl. tables"

Results with reference substance (positive control):

- Dose-response test: yes
- EC50: 24-hour: 1.6 mg/L (within the range given by the guideline: 24-hour EC50: 0.60-2.1 mg/L)

Reported statistics and error estimates:

The 48-hour EC50 and the 95% confidence limits were calculated by Weibull Analysis using linear maximum likelihood regression. Statistical analysis was performed using ToxRat Professional®. The 48-hour NOEC, EC0 and EC100 were determined directly from the raw data.

At the end of the two renewal periods, the test item concentrations were in the range of 89 to 101 % of the initially measured values. Thus, the concentrations of the test item were stable during the test medium renewal periods of 24 hours. The mean measured test item concentrations over the test period of 48 hours were calculated as the arithmetic mean of the test item concentrations measured at the start and the end of the two test medium renewal periods.

Table 1: Measured test item concentrations in the dilutions 1:2.2, 1:4.6, 1:10 and 1:22 at the start and end of the two test medium renewal periods

Dilution*	Measured Test Item Concentrations at the Start of the Renewal Periods (Day 0 / Day 1 )	Measured Test Item Concentrations at the End of the Renewal Periods ( Day 1 / Day 2 )	Mean Measured Concentration of the Test Item (arithmetic mean)
	[mg/L]	[mg/L]	[mg/L]
Dilution 1:46	n.a.	n.a.	n.a.
Dilution 1:22	0.88 / 0.91	0.78 / 0.90	0.87
Dilution 1:10	1.9 / 2.1	1.9 / 2.0	2.0
Dilution 1:4.6	4.4 / 4.6	4.2 / 4.6	4.4
Dilution 1:2.2	9.4 / n.a.°	8.4 / n.a.°	8.9

*: Dilution of the undiluted filtrate with a loading rate of 100 mg/L

n.a.: Not analyzed since below the NOEC determined in this test

n.a.°: Not analyzed since all daphnids were dead after 24 hours

 Table 2: Effect of the test item on the mobility of Daphnia magna

Dilution*	Mean Measured Concentration	No. of daphnids tested	Immobilized daphnids after 24 Hours		Immobilized daphnids after 48 Hours
	[mg/L]		No.	[%]	No.	[%]
Control	---	20	0	0	0	0
Dilution 1:46	n.a.	20	0	0	0	0
Dilution 1:22	0.87	20	0	0	0	0
Dilution 1:10	2.0	20	2	10	8	40
Dilution 1:4.6	4.4	20	11	55	20#	100
Dilution 1:2.2	8.9	20	20#	100	-	-

n.a.: Not analyzed since below the NOEC determined in this test

*: Dilution of the undiluted filtrate with a loading rate of 100 mg/L

#: Dead daphnids

Validity criteria fulfilled:

yes

Conclusions:

The test item had acute toxic effects on Daphnia magna in a 48-hour semi-static test according to OECD 202 and GLP. The 48-hour EC50 was calculated to be 2.1 mg/L with 95%-confidence limits of 1.8 and 2.9 mg/L.

Description of key information

EC50 (48h) = 2.1 mg/L (OECD 202, Daphnia magna, based on arithmetic mean measured concentration)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

2.1 mg/L

Additional information

One experimental study is available investigating the short-term effects of the substance to aquatic invertebrates. The acute toxicity of the test item to Daphnia magna was determined in a 48-hour test according to the OECD 202 (GLP). As the test item is a volatile substance, the test was performed using glass tubes completely filled with test medium (without headspace) that were tightly sealed with glass stoppers to avoid losses of test item by evaporation (closed system). A semi-static test design with a test medium renewal after 24 hours was used. Due to the low water solubility of the test item, the test media were prepared following the filtration method: The test item was mixed in test water at a loading rate of 100 mg/L, and was intensively stirred for 3 hours in a closed vessel. After stirring the emulsion was filtered through a 0.45 µm membrane filter. The dilutions 1:2.2, 1:4.6, 1:10, 1:22 and 1:46 of the undiluted filtrate were used as test media. At the end of the two renewal periods, the test item concentrations were in the range of 89 to 101 % of the initially measured values. Thus, the concentrations of the substance were stable during the test medium renewal periods of 24 hours. Immobilisation of D. magna was observed resulting in an EC50 (48 h) of 2.1 mg/L (based on arithmetic mean measured concentration).