1-Butoxy-4-[4-(trans-4-ethylcyclohexyl)-1-cyclohexen-1-yl]-2,3-difluorobenzene

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:

Jun 16 - Aug 20, 2020

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)

Principles of method if other than guideline:

A test was performed with 10 mg/L instead of 100 mg /L. The concentration was well above the water solubility of the substance.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

At the start of the exposure and at the renewal (0 and 24 hours), samples of the prepared test item concentrations were taken and analyzed. At the renewal and at the end of the exposure (24 and 48 hours), samples of the 24-hours old media were taken from additional replicates, which were prepared with test media, but without daphnids. These replicates were incubated under test conditions until sampling.

Vehicle:

no

Details on test solutions:

Culture Medium Elendt M4

Test organisms (species):

Daphnia magna

Details on test organisms:

Test system Daphnia magna STRAUS, obtained from continuous laboratory cultures.
Reason for the selection Daphnia magna is the preferred species in accordance with the
of the test system test guideline and is bred at the test facility.
Origin Institut für Wasser-, Boden- und Lufthygiene (WaBoLu), 14195 Berlin, Germany
Breeder Noack Laboratorien GmbH, Käthe-Paulus-Str. 1, 31157 Sarstedt, Germany
Culture In glass vessels (2 - 3 L capacity) with approximately 1.8 L culture medium, at 20 +/- 2 °C, 16 hours illumination; light intensity of max. 1500 lx
Culture medium Elendt M4, according to OECD 202, Annex 3 (2004), is used. .

Feeding of the culture stocks
The daphnids are fed at least 5 times per week ad libitum with a mix of unicellular green algae, e.g. Pseudokirchneriella subcapitata and Desmodesmus subspicatus. The algae are cultured at the test facility.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

261 mg/Ca CaO3/L (0 h)
271 mg/Ca CaO3/L (24 h)

Test temperature:

20.9 - 21.2 °C

pH:

7.8-8.1

Dissolved oxygen:

8.5 mg/L

Conductivity:

718 µS/cm (0 h)
708 µS/cm (0 h)

Nominal and measured concentrations:

nominal: 19.8 - 29.6 - 44.4 - 66.7 - 100% of the saturated solution
time weighted mean measured concentration: 72.9 - 142 - 192 - 311 - 598 µg/L

Details on test conditions:

Preparation of the saturated solution
A saturated solution with a nominal concentration of 10.0 mg test item/L was prepared once 96 ± 1 hour prior to the start of the exposure (at day -4). An appropriate amount of the test item was weighed out and transferred into a glass flask with an appropriate amount of dilution water. The saturated solution was stirred with a magnetic stirrer at 1100 rpm for 96 ± 1 hour at room temperature. Thereafter, the water phase was removed by siphoning from the middle of the glass flask. The first 25 mL were discarded. The saturated solution was checked via laser beam (Tyndall effect) for undissolved test item. The saturated solution showed a slight positive Tyndall effect. It was used as the highest loading rate and as a stock solution for the preparation of further dilution levels by diluting with dilution water. The mixing regime was selected based on the observations during the preliminary testing.

Test concentrations
Five concentrations of the test item with a separation factor of 1.5, prepared by diluting the saturated solution with dilution water (see Table 2), were tested as follows:
19.8 - 29.6 - 44.4 - 66.7 - 100% of the saturated solution
The test item concentrations were selected based on the results of a non-GLP preliminary range finding test. For results, see Annex II.

Control
Dilution water without test item incubated under the same conditions as the test groups

Reference Test
A reference test with potassium dichromate was conducted as an acute immobilization test (acc. to AQS P 9/2 and OECD 202) in Elendt M4 medium under static conditions with a test duration of 24 hours once per month in order to prove the validity of the test system and test conditions at the test facility.

Test method
The study was performed under semi-static conditions (with a renewal of the test solutions after 24 hours).

Test duration 48 hours

Test vessels Glass beakers (4 (ID) x 7 (H) cm), 50 mL capacity, loosely covered with watch glasses
Test volume 20 mL
Dilution water Same composition as the culture medium (see Table 2)

Number of daphnids and replicates
20 daphnids, divided into 4 replicates, each with 5 daphnids, were used for each concentration level and the control.

Age of the daphnids at the start of the exposure
Less than 24 hours old daphnids from a healthy stock were used for the study. To achieve this, juvenile daphnids were removed from the culture vessels at the latest 24 hours before the start of the exposure and discarded. The juveniles born within the following period were used for the test. No first brood progeny was used for the test.

Acclimatization
Acclimatization of the daphnids was not necessary, because the composition of the dilution water was equivalent to the culture medium.

Application
20 g test solution per replicate were weighed out into each test vessel. This corresponds to 20 mL per test vessel. The daphnids were inserted with a small amount of dilution water (start of the exposure) or test solution (water renewal) by pipette.

Renewal of the test solutions
The test solutions were renewed after 24 hours. For this purpose, a second set of test vessels was filled with the freshly prepared test solutions and the daphnids were transferred by pipette (see ‘Application’).
Test temperature (target) 18 - 22 °C, constant within ± 1 °C
Illumination (target) Diffuse light, light intensity of max. 1500 lx
Photoperiod (target) 16/8 hours light/dark cycle
Feeding The daphnids were not fed during the study.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 598 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: no effects detected up to the solubility limit

Details on results:

The saturated solution was visually clear and showed a slight positive Tyndall effect. The further concentration levels (19.8 to 100% of the saturated solution) were visually clear throughout the exposure.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
EC50 = 2.03 mg/L (1.00 - 4.00 mg/L)

Analytical Results:

Sampling data	Fresh media, 0 hours	Old media, 24 hours	Fresh media, 24 hours	Old media, 48 hours	Time weighted measured concentration [µg/L]
Nominal loading rate of the saturated solution of the test item [%]	Test Material
	Meas. conc. [µg/L]	Meas. conc. [µg/L]	Meas. conc. [µg/L]	Meas. conc. [µg/L]
100.0	1753	224	557	364	598
66.7	964	168	208	130	311
44.4	619	89.3	152	76.2	192
29.6	450	86.1	86.4	44.9	142
19.8	280	32.7	51.7	16.2	72.9
Control	< LOQ	< LOQ	< LOQ	< LOQ	< LOQ

Biological Results:

Nominal loading rate of the saturated solution of the test item / [%]	Geometric mean measured test item concentration / [µg/L]	Immobilsation
		absolute (after 24 h)	[%] after 24 h	absolute (after 48 h)	[%] after 48 h
100.0	598	0 / 20	0	0 / 20	0
66.7	311	0 / 20	0	0 / 20	0
44.4	192	0 /20	0	0 / 20	0
29.6	142	0 / 20	0	0 / 20	0
19.8	72.9	0 / 20	0	0 / 20	0
Control		0 / 20	0	0 / 20	0

Validity criteria fulfilled:

yes

Conclusions:

The EC50 value of the test material is thus clearly above the water solubility of the pure substance corresponding to a nominal concentration of 100 mg test item/L.

Executive summary:

In the acute immobilization test with Daphnia magna (STRAUS), the effects of the test item were determined at the test facility according to OECD 202 (2004).
The study was conducted under semi-static conditions over a period of 48 hours with five dilution levels of a saturated solution of the test item (nominal: 19.8 – 29.6 – 44.4 – 66.7 – 100% of the saturated solution) prepared in a geometric series with a separation factor of 1.5 between the dilution levels.
The saturated solution was visually clear and showed a slight positive Tyndall effect.The further concentration levels were visually clear.
Twenty daphnids (divided into 4 replicates with 5 daphnids each) were exposed to each concentration level and the control.
The concentrations of the test item were analytically verified via GC-MS in the fresh media of all concentration levels and in the control at the start of the exposure and at the renewal (0 and 24 hours) as well as in the 24-hours old media at the renewal and at the end of the test (24 and 48 hours).
The measured concentrations were in the range of 12 to 19% of the initially measured concentrations in the old media for the first interval and in the range of 31 to 65% of the initially measured concentrations in the old media for the second interval.The geometric mean measured concentrations of the test material are 72.9 – 142 – 192 – 311 – 598 µg/L.
No immobilisation or any other biological effect was noted in the course of this study.

The EC50 value of the test material is thus clearly above the water solubility of the pure substance corresponding to a nominal concentration of 100 mg test item/L.

Description of key information

In the OECD 202 assay performed for this compound, no effects on Daphnia magna were observed up to the saturated solution of the test item.

Based on the time weighted mean measured concentrations of the test substance, the 48 hour EC50 for Daphnia magna was > 598 µg/L corresponding to a nominal concentration of 100 mg/L.

Additional information

In the acute immobilization test with Daphnia magna (STRAUS), the effects of the test item were determined at the test facility according to OECD 202 (2004).
The study was conducted under semi-static conditions over a period of 48 hours with five dilution levels of a saturated solution of the test item (nominal: 19.8 – 29.6 – 44.4 – 66.7 – 100% of the saturated solution) prepared in a geometric series with a separation factor of 1.5 between the dilution levels.
The saturated solution was visually clear and showed a slight positive Tyndall effect.The further concentration levels were visually clear.
Twenty daphnids (divided into 4 replicates with 5 daphnids each) were exposed to each concentration level and the control.
The concentrations of the test item were analytically verified via GC-MS in the fresh media of all concentration levels and in the control at the start of the exposure and at the renewal (0 and 24 hours) as well as in the 24-hours old media at the renewal and at the end of the test (24 and 48 hours).
The measured concentrations were in the range of 12 to 19% of the initially measured concentrations in the old media for the first interval and in the range of 31 to 65% of the initially measured concentrations in the old media for the second interval.The geometric mean measured concentrations of the test material are 72.9 – 142 – 192 – 311 – 598 µg/L.
No immobilisation or any other biological effect was noted in the course of this study.

The EC50 value of the test material is thus clearly above the water solubility of the pure substance corresponding to a nominal concentration of 100 mg test item/L.