(R)-5-isopropyl-2-methylcyclohexa-1,3-diene

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:

2017-08-29 to 2017-09-01

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

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Sampling method: At the start of the exposure and the renewal (0 and 24 hours), samples of the fresh media were taken after preparation of all test item concentrations 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 directly from the test vessels and analyzed.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A saturated solution with a nominal concentration of 4.37 mg/L of the test item, which corresponds to 5.218 µL/L of the test item (relative density: 0.8375 (D20/4) was taken into account), was prepared one day prior the start of exposure (at -24 hours) and one day prior to the renewal of the test solutions (at o hours) in a brown glass flask. The glass flask was filled with 40 g glass beads (diameter: 4 mm) and up to the top with the dilution water having no headspace (total volume: 2275 mL). The test item was pipetted into the dilution water and the brown glass flask was closed immediately with a screw cap with a septum. This dispersion was shaken for 24 hours with 20 rpm at room temperature. After completion of shaking, the dispersion was allowed to stand for approximately 1 hour for separation of undissolved test item. Thereafter, the saturated solution was removed from the approximate center of the water body. The saturated solution was used as the highest concentration level and as a stock solution for the preparation of further dilution levels by dilution with dilution water.
- Differential loading: Five dilution leves were prepared out of the sturated solution in a geometric series with a separation factor of 2.2 by dilution of the saturated solution with dilution water, were tested as follows: 1.94 - 4.27 -9.93 - 20.7 - 45.5 - 100% of the saturated solution.
- Controls: Negative control = dilution water without test item incubated under the same conditions as the test groups; positive control with the reference item potassium dichromate
- Evidence of undissolved material: The saturated solution was checked via laser beam (Tyndall effect) for undissolved test item (formation of an emulsion). The Tyndall effect was positive.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea
- Strain/clone: Clone 5
- Breeder: Boack Laboratorien GmbH, Käthe-Paulus-Str. 1, 31157 Sarstedt, Germany
- Origin: Institut für Wasser-, Boden- und Lufthygiene (WaBoLu), 14195 Berlin, Germany
- Age of parental stock: Less than 24 hours old daphnids from a healthy stock were used for the study. Juvenile daphnids were removed from the culture vessels at the latest 24 hours before the start of the exposure and discarded. The juvenile born within the following period of max. 24 hours preceding the exposure were used for the test. No first brood progeny was used for the test.
- Feeding during test: None

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

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

No

Hardness:

177- 180 mg CaCO3/L (dilution water)

Test temperature:

18- 22 +- 1 °C

pH:

7.45 - 8.62

Dissolved oxygen:

7.21 - 8.69 mg/L

Conductivity:

432 - 482 µS/cm (dilution water)

Nominal and measured concentrations:

Nominal concentration of saturated solution [mg/L]: 1.94, 4.27, 9.39, 20.7, 45.5, 100% of the saturated solution and control
Geometric mean measured test item concentration [mg/L]: 0.042, 0.104, 0.207, 0.452, 1.043, 2.131, 0 (control)

Details on test conditions:

TEST SYSTEM
- Test vessel: Flask
- Material, size, headspace, fill volume: Glass, 4.5 (ID) x 9.5 (H) cm, with screw, filled up to the top with the test solutions (130 mL)
- Aeration: No
- No. of organisms per vessel: 5
- No. of vessels per concentration: 4
- No. of vessels per control: 4
- Renewal rate of test solution: Once after 24 hours of exposure

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16/8 hours light/dark cycle
- Light intensity: Diffuse light, light intensity of max. 1500 lux

EFFECT PARAMETERS MEASURED: Mobility (after 24 and 48 hours). An animal was considered immobile, if it was not able to swim in the water phase within 15 seconds after gentle aggitation of the test vessel.

RANGE-FINDING STUDY
- Test concentrations: Yes
- Results used to determine the conditions for the definitive study: Yes

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

513 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mobility

Remarks on result:

other: 95% confidence limits: 421 - 717 µg/L

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

280 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mobility

Remarks on result:

other: 95% confidence limits: 153 - 392 µg/L

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

2 131 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mobility

Duration:

24 h

Dose descriptor:

EC10

Effect conc.:

863 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mobility

Remarks on result:

other: 95% confidence limits: => 207 - 1715 µg/L

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

1 191 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mobility

Remarks on result:

other: 95% confidence limits: 1059 - 1861 µg/L

Duration:

24 h

Dose descriptor:

EC100

Effect conc.:

2 131 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mobility

Details on results:

- Other biological observations: Other adverse affects did not appear
- Mortality of control: No
- Other adverse effects control: No
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

EC50 = 1.90 mg/L (95% confidence limits: 1.43 - 2.69 mg/L)

Reported statistics and error estimates:

EC10 and EC50 values were calculated by sigmoidal dose-response regression with the software GraphPad Prism. The respective 95% confidence limits were calculated from the standard error and the t-distribution.

Table 1: Immobilization rates after 24 and 48 hours of Exposure in the Definitive Test (n = 20, divided into 4 replicates with 5 daphnids each)

Geometric mean measured concentration of the test item [µg/L]	IMMOBILIZATION [%]
	Replicates					Replicates
	1	2	3	4	MV	1	2	3	4	MV
2131	100	100	100	100	100	100	100	100	100	100
1043	20	40	0	40	25	60	100	100	100	90
452	0	60	40	0	25	20	80	60	0	40
207	0	0	0	0	0	0	0	0	0	0
104	0	0	0	0	0	0	0	0	0	0
42	0	0	0	0	0	0	0	0	0	0
Control	0	0	0	0	0	0	0	0	0	0

Table 2: Measured Concentrations of the Test Item during the Definitive Test.

Sampling date	Fresh media, 0 hours	Old media, 24 hours		Fresh media, 24 hours	Old media, 48 hours		Geometric mean measured test item concentration [µg/L]
Dilution level of the saturated solution [%]	PHELLANDREN FRACTION EX EUCALYPTUS OIL
	Meas. conc. [µg/L]	Meas. conc. [µg/L]	%	Meas. conc. [µg/L]	Meas. conc. [µg/L]	%
100*	2456	1850	75	3156 ^1)	-	-	2131
45.5	1088	771	71	1368	1031	75	1043
20.7	490	366	75	569	409	72	452
9.39	242	160	66	272	173	63	207
4.27	117	73.9	63	141	94.3	67	104
1.94	46.9	27.6	59	60.4	39.7	66	42
Control	< LOQ	< LOQ		< LOQ	< LOQ

Meas. conc. = measured concentration of the test item, enrichment and dilution factors taken into account

% = percentage of the initially measured concentration of the test item

LOQ = limit of quantification

¹⁾ = saturated solution analyzed only on start of the exposure, but not taken into account for calculation of the geometric mean measured concentration, due to 100% mortality after 24 hours

* = saturated solution

Validity criteria fulfilled:

yes

Conclusions:

In a valid study according to OECD Guideline 202 and EU Method C.2, the 48h EC50 of the test item was determined to be 513.0 µg/L (based on geometric mean measured concentration).

Executive summary:

To assess the ecotoxicological effects of the test item to aquatic invertebrates, an Acute Toxicity Test with Daphnia magna according to OECD Guideline 202 and EU Method C.2 was carried out in compliance with GLP principles. The study was conducted in a closed system (sealed glass flasks) without headspace under semi-static conditions over a period of 48 hours with the undiluted saturated solution of the test item and further five dilution levels (nominal 1.95 to 100%) prepared from the saturated solution in a geometric series with a separation factor of 2.2. Twenty daphnids (devided 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 at the start of the exposure and at the renewal of the test solutions (0 and 24 hours) as well as in the old media at the renewal and at the end of the test (24 and 48 hours) in all concentration levels and in the control. The geometric mean measured test item concentration was 2131, 1043, 452, 207, 104, 42.0 µg/L. In result, the 48h EC10 and EC50 of the test item was determined to be 0.280 mg/L and 0.513 mg/L (based on geometric mean measured concentration), respectively. All validity criteria of the guidelines were fulfilled.

Description of key information

The 48h EC50 of the substance was determined to be 0.513 mg/L (based on geometric mean measured concentration).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.513 mg/L

Additional information

To assess the ecotoxicological effects of the test item to aquatic invertebrates, an Acute Toxicity Test with Daphnia magna according to OECD Guideline 202 and EU Method C.2 was carried out in compliance with GLP principles. The study was conducted in a closed system (sealed glass flasks) without headspace under semi-static conditions over a period of 48 hours with the undiluted saturated solution of the test item and further five dilution levels (nominal 1.95 to 100%) prepared from the saturated solution in a geometric series with a separation factor of 2.2. Twenty daphnids (devided 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 at the start of the exposure and at the renewal of the test solutions (0 and 24 hours) as well as in the old media at the renewal and at the end of the test (24 and 48 hours) in all concentration levels and in the control. The geometric mean measured test item concentration was 2131, 1043, 452, 207, 104, 42.0 µg/L. In result, the 48h EC10 and EC50 of the test item was determined to be 0.280 mg/L and 0.513 mg/L (based on geometric mean measured concentration), respectively. All validity criteria of the guidelines were fulfilled.