reaction mass of [(1R*,5S*)-3,3,5-trimethylcyclohexyl] acetate and [(1S*,5S*)-3,3,5-trimethylcyclohexyl] acetate

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:

03 Mar - 06 Mar 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)

GLP compliance:

yes (incl. QA statement)

Remarks:

Staatliches Gewerbeaufsichtsamt Hildesheim, Hildesheim, Germany

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 6.25, 12.5, 25.0, 50.0 and 100% of a saturated stock solution (analysed at 0, 24 and 48 h)
- Sample storage conditions before analysis: Samples were analysed directly.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A stock solution of 40 mg/L was freshly prepared before start of each exposure (at 0 and 24 h). 132 µL test item was directly introduced by pipette into a 3 L measuring flask and completely filled with the dilution water. The stock solution was stirred at approx. 1100 rpm for 2 h at room temperature.
- Eluate: no
- Differential loading: no
- Controls: yes, test medium control
- Evidence of undissolved material: Presence of undissolved test item during preparation of test concentrations and during the test was not observed.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: water flea
- Strain: Clone 5
- Age at study initiation (mean and range, SD): 2-24 h
- Method of breeding: Daphnids were bred at the laboratory in Elendt M4 medium modified to a total hardness of 160 - 180 mg CaCO3/L. Daphnids were cultured in glass vessels (2-3 L) with approx. 1.8 L of medium at 20 ± 2 °C, 16 h of illumination and a light intensity of max. 20 µE*m-2*s-1. Feeding was done at least 5 times per week ad libitum with a mix of unicellular algae with an algae cell density of > 10E+06 cells/mL. The algae were cultured at test facility.
- Feeding during test: none

ACCLIMATION
- Acclimation period: at least 2 h
- Acclimation conditions: same as test
- Type and amount of food:a mix of unicellular green algae, e.g. Pseudokirchneriella subcapitata and Desmodesmus subspicatus, with an algae cell density of > 10^6 cells/mL.
- Feeding frequency: at least 5 times per week

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

265 - 269 mg CaCO3/L

Test temperature:

19.8 - 20.2 °C

pH:

7.11 - 7.50

Dissolved oxygen:

6.70 - 9.03 mg/L

Nominal and measured concentrations:

nominal: control, 6.25, 12.5, 25.0, 50.0 and 100% of a saturated stock solution
measured: < LOQ, 2.00, 4.30, 6.83, 15.0, 27.8 mg/L (geometric mean)

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass flasks
- Type (delete if not applicable): sealed with screw caps
- Material, size, headspace: glass, size: 4.5 (IC) x 9.5 (H) cm, ca. 130 mL, headspace: nearly no headspace
- Aeration: no
- Renewal rate of test solution (frequency/flow rate): every 24 h (freshly prepared test solutions were filled in a second set of test vessels and the daphnids were transferred with a pipette)
- 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: according to guideline
- Culture medium different from test medium: no
- Intervals of water quality measurement: after 0, 24 and 48 h

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h light/8 h dark
- Light intensity: diffuse light (max. 20 µE*m-2*s-1)

EFFECT PARAMETERS MEASURED: mobility after 24 h

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study
- Test concentrations: 0.125, 1.36, 13.8 mg/L (measured; 1st range-finding study), 0.231, 2.49, 23.0 mg/L (measured, 2nd range-finding study)
- Results used to determine the conditions for the definitive study: 1st range-finding study: 15% immobilisation at 13.8 mg/L, 5% immobiliation at 1.36 mg/L. The lowest concentration was discarded; 2nd range-finding study: 70% immobilisation at 23.0 mg/L, 5% immobilisation at 2.49 mg/L. The lowest concentration was discarded.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

15 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

22 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CI: 16.0 - 27.7 mg/L

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

> 27.8 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

20.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CI: 15.1 - 27.6 mg/L

Details on results:

- Mortality of control: 0%
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50/LC50: EC50 (48 h): 1.51 mg/L (95% CI 1.08 - 2.10 mg/L)

Reported statistics and error estimates:

The ECx values were statistically calculated from a sigmoidal dose-response curve with variable slope. The 95 % confidence limits were calculated from the best-fit values, the standard error and the t-distribution with the software GraphPad prism 5. The concentration effect relationships are shown graphically. The EC50-value for the reference item and its 95 % confidence limits were calculated accordingly.

The measured concentrations of the test item at the start of the exposure intervals (0 and 24 h) were in the range of 68 to 93% of nominal. At the end of the exposure intervals (48 h), the measured concentrations of the test item were in the range of 57 to 85% of nominal and 77 to 115 % of the initially measured concentrations. The measured test item concentrations did not remain within ± 20 % of the nominal loadings or the initially measured concentrations. Thus, the effect concentrations were based on the geometric mean measured concentrations.

Table 1: Immobilisation rates after 24 and 48 h of exposure in the definitive test

Test item		Immobilisation [%]
Nominal concentration [mg/L]	Geometric mean measured concentration [mg/L]	24 h					48 h
		Replicates					Replicates
		1	2	3	4	Mean	1	2	3	4	Mean
40	27.8	80	80	100	60	80	100	100	100	60	90
20	15.0	0	0	0	0	0	0	0	0	0	0
10	6.83	0	0	0	0	0	0	0	0	0	0
5	4.30	0	0	0	0	0	0	0	0	0	0
2.50	2.00	0	0	0	0	0	0	0	0	0	0
Control	<LOQ	0	0	0	0	0	0	0	0	0	0

Validity criteria fulfilled:

yes

Conclusions:

This study resulted in a EC50 (48 h) of 22.0 mg/L (geom. mean measured concentration) based on mobility of D. magna (OECD 202, GLP).

Description of key information

EC50 (48 h): 22.0 mg/L based on mobility of D. magna (OECD 202)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

22 mg/L

Additional information

One experimental study is available investigating the effects of the substance to aquatic invertebrates. The study was performed according to OECD 202 (GLP) under semi-static conditions using Daphnia magna as test organism. Nominal test concentrations of 6.25, 12.5, 25.0, 50.0 and 100% of a saturated stock solution as well as a control were tested. The actual exposure concentrations were verified by GC/FID analysis at the beginning and after 24 and 48 h of the study. Measured test item concentrations did not remain within ± 20% of the nominal concentrations. Thus, the effect concentrations were based on the geometric mean of the measured concentrations. At the end of the study an EC50 (48 h) of 22.0 mg/L (95% CI: 16.0 - 27.7 mg/L) was calculated based on mobility.