[1R-(1α,2β,5α)]-1-(isopropyl)-2-methoxy-4-methylcyclohexane

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:

13 Jul - 16 Jul 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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Staatliches Gewerbeaufsichtsamt, Hildesheim, Germany

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 1.25, 2.5, 5, 10, 20 mg/L (nominal)
- Sampling method: all concentration levels and controls were analytically verified. At the start of the exposure intervals (0 and 24 hours), sampling was carried out after preparation of the test concentrations. At the end of the exposure intervals (24 and 48 hours), samples were taken directly from the test vessels
- Sample storage conditions before analysis: all samples were stored at room temperature before preparation. Prepared samples were then stored at room temperature until analysis

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution of 20.0 mg/L of the test item was prepared one day before the start of each exposure interval (at -24 and 0 hours). For this purpose, 48 µL of the test item (density of the test item taken into account) were directly introduced by pipette into a 2 L measuring flask, completely filled with the dilution water below the water surface while stirring. The stock solution was stirred with approximately 1100 rpm for 24 hours at room temperature. Due to the volatile test item properties, a semi-static test design with a daily renewal of the test solutions in glass flasks sealed with screw caps (made from polypropylene) was chosen to reduce contact with air and losses of the test item. The glass flasks were filled up with the test solutions having nearly no headspace.
- Controls: dilution water without test item tested under the same conditions as the test groups

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: water flea
- Age at study initiation: 2 - 24 hours old daphnids, juvenile daphnids were removed from culture vessels latest 22 hours before the start of the exposure. The juveniles born within this period of max. 22 hours were used for the test after an acclimatisation phase in the dilution water of at least 2 hours. No first brood progeny was used for the test
- Method of breeding: culture medium was Elendt M4, according to Elendt (1990), modified to a total hardness of 160 to 180 mg CaCO3/L. Culture was grown in glass vessels (2 - 3 L capacity) with approximately 1.8 L culture medium, at 20 ± 2 °C, in an incubator, 16 h illumination, light intensity of max. 20 µE x m^-2 x s^-1. Daphnids were fed with ad libitum with a mix of unicellular green algae, e.g. Pseudokirchneriella subcapitata and Desmodesmus subspicatus, with an algae cell density of > 106 cells/mL, at least 5 times per week.
- Feeding during test: no

ACCLIMATION
- Acclimation period: at least 2 hours in dilution water

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

0 h: 272 mg CaCO3/L
24 h: 263 mg CaCO3/L

Test temperature:

0 h: 20.5 °C
24 h: 20.7 °C

pH:

pH at the start of the exposure: at 0 h the pH was 7.51 - 7.95, at 24 h the pH was 7.65 - 7.83
pH at the end of the exposure: after 24 h the pH was 7.49 - 9.14, after 48 h the pH was 7.53 - 7.63

Dissolved oxygen:

at the start of the exposure: at 0 h dissolved oxygen was 9.10 - 9.75 mg/L and at 24 h 9.24 - 10.03 mg/L
at the end of the exposure: after 24 h dissolved oxygen was 7.50 - 9.70 mg/L and after 48 h 7.53 - 7.63 mg/L

Nominal and measured concentrations:

- Nominal test item concentrations: 20, 10, 5, 2.5, 1.25 mg/L and control (concentrations selected based on the results of the non GLP preliminary range finding tests)
- Geometric mean measured concentrations: 12.4, 6.88, 3.84, 2.10, 0.963 mg/L and control

Details on test conditions:

TEST SYSTEM
- Test vessel: sealed glass flasks (4.5 (ID) x 9.5 (H) cm, ca. 130 mL test volume without headspace per concentration and control) with screw caps made from polypropylene
- Type: closed
- Renewal 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
- Concentrations of reference substance: 1.00-2.00-4.00 mg/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to guideline
- mg CaCO3/L: 272 mg CaCO3/L at 0 h and 263 mg CaCO3/L at 24 h
- Conductivity: 651 µS/cm at 0 h and 640 µS/cm at 24 h
- Culture medium different from test medium: yes, culture medium according to Elerndt, 1990 (Elendt B. P., 1990: Selenium Deficiency in Crustacea, Protoplasma 154, 25 - 30)
- Intervals of water quality measurement: at the start (0 and 24 h) and at the end (24 and 48 h) of the exposure time

OTHER TEST CONDITIONS
- Photoperiod: 16/8 h light/dark cycle
- Light intensity: diffuse light, light intensity of max. 20 µE x m^-2 x s^-1 (max. 1340 lx)

TEST CONCENTRATIONS
- Range finding study: yes
- Test concentrations: 0.200 - 2.00 - 20.0 mg/L (nominal concentrations)
- Results used to determine the conditions for the definitive study: see table 1 in `any other information on materials and methods´

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

6.44 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CI: 6.29 - 6.58 mg/L

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

3.84 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Behavioural abnormalities: no
- Other biological observations: no
- Mortality of control: no

Results with reference substance (positive control):

- Results with reference substance valid: yes
- EC50: 1.88 mg/L (95% Cl: 1.62-2.19 mg/L) (within the recommended range of 0.6 - 2.1 mg/L according to guideline)
- Other: reference item was determined after 24 hours

Reported statistics and error estimates:

EC10- and EC50-values (after 24 and 48 hours) were calculated by sigmoidal dose-response regression. 95 % confidence limits were calculated from the standard error and the t-distribution. All calculations were carried out from the best-fit values with the software GraphPad Prism5. The EC50-value for the reference item and its 95 % confidence limits were calculated accordingly.

The EC100-values (after 24 and 48 hours) and the NOEC and LOEC (after 48 hours) were deduced directly from the observation data, without mathematical calculation. All effect levels (EC10 / 50 /100, NOEC and LOEC) were based on the geometric mean measured concentrations of the test item, as measured test item concentrations did not remain within ± 20 % of the nominal concentrations.

Table 2: EC10-, EC50- (with 95 % Confidence Limits), EC100-values, NOEC and LOEC

Effect levels	Test duration [h]	Geometric mean measured test item concentrations [mg/L]
EC10 (with 95 % confidence limits)	24	6.39 (Cl: 6.04 - 6.79)
	48	6.12 (Cl: 6.00-6.38)
EC50 (with 95 % confidence limits)	24	7.83 (Cl: 7.46 - 8.22)
	48	6.44 (Cl: 6.29 - 6.58)
EC100	24	> 12.4
	48	12.4
NOEC	48	3.84
LOEC	48	6.88

Table 3: Immobilisation rates after 24 and 48 h of exposure

		IMMOBILISATION [%]
Nominal test item concentration [mg/L]	Geometric mean measured concentration [mg/L]	24 hours					48 hours
		Replicates					Replicates
		1	2	3	4	MV	1	2	3	4	MV
20	12.4	100	100	100	80	95	100	100	100	100	100
10	6.88	0	20	40	20	20	80	100	100	100	95
5	3.84	0	0	0	0	0	0	0	0	0	0
2.5	2.1	0	0	0	0	0	0	0	0	0	0
1.25	0.963	0	0	0	0	0	0	0	0	0	0
Control		0	0	0	0	0	0	0	0	0	0

Table 4: Measured concentrations and % of the nominal concentrations of the test Item

during the test

Sampling	0 h (start of the exposure)		24 h (end of the exposure)		24 h (start of the exposure)		48 h (end of the exposure)		Geometric mean
Nominal test item concentration [mg/L]	Meas. con. [mg/L]	%	Meas. con. [mg/L]	%	Meas. con. [mg/L]	%	Meas. con. [mg/L]	%	Meas. con. [mg/L]	%
20	13.4	67	7.9	40	15.7	79	14.2	71	12.4	62
10	8.5	85	4.8	48	8.41	84	6.52	65	6.88	69
5	4	80	3.12	62	4.65	93	3.74	75	3.84	77
2.5	2.22	89	1.87	75	2.49	100	1.89	76	2.1	84
1.25	0.973	78	0.828	66	1.34	107	0.797	64	0.963	77
Control	< LOQ		< LOQ		< LOQ		< LOQ

Validity criteria fulfilled:

yes

Conclusions:

The test substance caused immobilisation of Daphnia magna in a guideline study according to OECD 202 (GLP). An EC50 (48 h) of 6.44 mg/L based on the geometric mean measured concentration was calculated.

Description of key information

EC50 (48 h) Daphnia magna = 6.44 mg/L (geometric mean measured, OECD 202)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

6.44 mg/L

Additional information

One study is available, in which the short term toxicity to invertebrates was investigated according to GLP and OECD guideline 202. In a semi-static test system in freshwater, the test organism Daphnia magna was exposed to five nominal concentrations of 1.25, 2.50, 5.00, 10.0, and 20.0 mg/L. The test was conducted with closed bottles due to the volatility of the substance. The analytical samples were analysed at the start of the exposure intervals (0 and 24 hours) and at test end (24 and 48 hours).The measured concentrations were in the range of 67 to 107 % of the nominal concentrations at test start. At the end of the exposure intervals, the measured concentrations of the test item were in the range of 40 to 76 % of the nominal concentrations. Since the measured test item concentrations did not remain within ± 20 % of the nominal concentrations, the effect values are based on the geometric mean measured concentrations of the test item calculated as follows: 0.963, 2.10, 3.84, 6.88 and 12.4 mg/L. Based on the geometric mean measured concentrations of the test item, the 48 hour-EC50 for Daphnia magna was 6.44 mg/L (95 % confidence limits: 6.29 - 6.58 mg/L).