Reaction mass of (1RS)-1-[(1RS)-3,3-dimethylcyclohexyl]ethyl [(2RS,3RS)-3-ethyloxiran-2-yl]acetate and (1RS)-1-[(1RS)-3,3-dimethylcyclohexyl]ethyl [(2SR,3SR)-3-ethyloxiran-2-yl]acetate and (1RS)-1-[(1SR)-3,3-dimethylcyclohexyl]ethyl [(2RS,3RS)-3-ethyloxiran-2-yl]acetate and (1RS)-1-[(1SR)-3,3-dimethylcyclohexyl]ethyl [(2SR,3SR)-3-ethyloxiran-2-yl]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:

05-09-2014 to 20-02-2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP. All relevant validity criteria were met.

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

Principles of method if other than guideline:

An Acute Immobilisation Test to Daphnia magna STRAUS was carried out to determine the EC50-values of the test item after 24 and 48 hours of exposure under semi-static conditions in a closed system and in darkness.

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected June 2013; signature: November 2013

Analytical monitoring:

yes

Details on sampling:

- Concentrations: All relevant concentration levels and the control were analytically verified via GC-MS at the start (0 and 24 hours) and at the end of the exposure intervals (24 and 48 hours). Definitive Test: Nominal concentrations: 0 (control), 0.5, 1.0, 2.0, 4.0 and 8.0 mg/L ; equivalent geometric mean measured concentrations: 0 (control), 0.392, 0.86, 1.65, 2.79 and 6.56 mg/L
- Sampling method: 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. The method was validated prior to this study according to SANCO 3029/99 rev.4 (2000).
- Sample storage conditions before analysis: All original samples were stored at 6 ± 2 °C if necessary. Prepared samples were stored in an autosampler at room temperature until analysis.

Vehicle:

no

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphinds
- Strain: Daphnia magna STRAUS (Clone 5)
- Source: in-house laboratory cultures
- Age at study initiation (mean and range, SD): 2 to 24 hours old daphnids from a healthy stock were used for the study
- Method of breeding: Not reported.
- Feeding during test: No. The daphnids were not fed during the study. During culture: The culture 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, with an algae cell density of > 10^6 cells/mL. The algae are cultured at the test facility
- Food type: Not applicable.
- Amount: Not applicable.
- Frequency: Not applicable.

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:

Dilution water quality parameters: 0 hours: Total Hardness: 262 mg CaCO3/L ; 24 hours Total Hardness: 274 mg CaCO3/L

Test temperature:

Dilution water quality parameters: 0 hours: Temperature 19.8 °C ; 24 hours: Temperature 19.0 °C

pH:

Dilution water quality parameters: 0 hours: pH 7.62 ; 24 hours: pH 7.67; mean pH at 48 hours all replicates: did not differ by more than 0.2 units

Dissolved oxygen:

Dilution water quality parameters: 0 hours: O2 dissolved: > 8.34 mg/L ; 24 hours: O2 dissolved: > 8.31 mg/L; mean dissolved oxygen at 48 hours all replicates: did not decrease to less than 8.31 mg/L.

Nominal and measured concentrations:

Definitive test: Nominal concentrations: 0 (control), 0.5, 1.0, 2.0, 4.0 and 8.0 mg/L
Measured concentrations were determined and are available in the full study report.
Equivalent geometric mean measured concentrations: 0 (control), 0.392, 0.86, 1.65, 2.79 and 6.56 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 4 (ID) x 7 (H) cm, ca. 50 mL with 20 mL fill volume
- Type: A semi-static test design with a daily renewal of the test solutions in glass flasks loosely covered with watch glasses.
- Material, size, headspace, fill volume: Glass, ca. 50 mL with 20 mL fill volume, loosely covered with watch galsses.
- Aeration: No aeration of the test solutions.
- No. of organisms per vessel: Control and test item: 20 per concentration, 5 per vessel (divided into 4 replicates)
- No. of vessels per concentration (replicates): 4 (four replicates, 5 daphnia per vessel).
- No. of vessels per control (replicates): 4 (four replicates, 5 daphnia per vessel).
- No. of vessels per vehicle control (replicates): Not applicable.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ISO Test water, according to OECD 202, Annex 3: composition (mg/L): KCl 5.76; NaHCO3: 64.8; CaCl2.2H2O: 294 ; MgSO4.7H2O: 123.
- Culture medium different from test medium: Yes. Elendt M4, according to ELENDT (1990), modified to a total hardness of 160 to 180 mg CaCO3/L
Per replicate, 20 g test solution were weighed out into the 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 a pipette.

OTHER TEST CONDITIONS
- Adjustment of pH: No.
- Photoperiod: 16/8 h light/dark cycle during culture conditions. During testing: Light exclusion.
- Light intensity: Diffuse light, light intensity of max. 20 µE /m2 s (1340 lx)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Immobility (including mortality), 24 hours and at 48 hours. An organism was considered to be immobile, if it was not able to swim in the water phase within 15 seconds after gentle agitation of the test vessel. Other observations were not made.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.0
- Justification for using less concentrations than requested by guideline: Not applicable.
- Range finding study
- Test concentrations: Not reported.
- Results used to determine the conditions for the definitive study: Yes.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

4.33 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CL: 3.75 - 5.01 mg/L ; geometric mean measured concentrations

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

3.18 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CL: 2.91 - 3.93 mg/L; geometric mean measured concentrations

Details on results:

- Behavioural abnormalities: None reported.
- Observations on body length and weight: Not applicable.
- Other biological observations:
- Mortality of control: No mortalities in control.
- Other adverse effects control: None reported.
- Abnormal responses: None reported.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None.
- Effect concentrations exceeding solubility of substance in test medium: No.

Results with reference substance (positive control):

- Results with reference substance valid?: Yes.
- Mortality: None. Acute immobilisation observed only.
- EC50/LC50: 24h-EC50 was 2.01 mg/L (C.I. 1.71 - 2.26).
- Other: The EC50-value of the reference item potassium dichromate after 24 hours is within the prescribed concentration range of 0.6 - 2.4 mg/L of quality criteria according to AQS P 9/2 (02/2000) listed in DIN 38412 - L 30 for daphnids clone 5 cultured in Elendt M4 medium. The EC50-value of the reference item is also within the recommended range of 0.6 - 2.1 mg/L according to OECD Guideline TG 202

Reported statistics and error estimates:

The EC50-value after 24 and 48 hours of exposure were calculated by sigmoidal dose-response regression. The respective 95% confidence limits were calculated from the standard error and the t-distribution.

Table 1. Immobilisation Rates after 24 and 48 h of Exposure in the Definitive Test

(n = 20, divided into 4 replicates with 5 daphnids each)

Geometric mean measured test item concentration [mg/L]	Immobilisation [%]
	24 hours					48 hours
	Replicates					Replicates
	1	2	3	4	MV	1	2	3	4	MV
6.56	60	60	80	100	75	80	100	100	100	95
2.79	20	20	20	40	25	60	20	20	40	35
1.65	0	0	0	0	0	0	0	0	0	0
0.860	0	0	0	0	0	0	0	0	0	0
0.392	0	0	0	0	0	0	0	0	0	0
Control	0	0	0	0	0	0	0	0	0	0

 

Table 3. Measured Concentrations and Percent of the Nominal and the Initially Measured Concentrations of the Test Item during the Definitive Test

Sampling	0 hours Start of the exposure interval		24 hours End of the exposure interval		24 hours Start of the exposure interval		48 hours End of the exposure interval		Geometric Mean Measured
Nominal test item concentration [mg/L]	Meas. conc. [mg/L]	%	Meas. conc. [mg/L]	%	Meas. conc. [mg/L]	%	Meas. conc. [mg/L]	%	Meas. conc. [mg/L]	%
8.00	6.43 #1	80	7.26	91	7.05	88	5.63	70	6.56	82
4.00	2.59 #2	65	3.72	93	2.54	64	2.48	62	2.79	70
2.00	1.96 #1	98	1.66	83	1.73	87	1.33	67	1.65	83
1.00	1.02 #1	102	0.895	90	0.852	85	0.704	70	0.860	86
0.50	0.358 #1	72	0.377	75	0.509	102	0.344	69	0.392	78
0.00 (Control)	< LOQ		< LOQ		< LOQ		< LOQ		< LOQ

#1 : Mean of two replicates

#2 : Mean of three replicates

LOQ = 0.1 mg test item / L

Validity criteria fulfilled:

yes

Conclusions:

The test item 48h-EC50 was 3.18 mg/L (95% CL: 2.91 – 3.93 mg/L) based on geometric mean measured concentrations.

Executive summary:

The acute toxicity to Daphnia magna was carried out according to OECD TG 202 Daphnia sp., Acute Immobilisation Test and EU Method C.2 guidelines under GLP. The study was conducted in a closed system (sealed glass flasks) without headspace under semi-static conditions and under light exclusion over a period of 48 hours with five concentration levels of the test item in the range of 2.50 to 40.0 mg/L prepared in a geometric series with a separation factor of 2. To reduce losses of the test item, the study was conducted under semi-static conditions in darkness and in a closed system. A stock solution of 40.0 mg/L was prepared with ISO test water (according to OECD 202, Annex 3) which was adjusted to a pH of 6 ± 0.2 by addition of 1 M HCI prior to testing, with a magnetic stirrer at approximately 1100 rpm and was used as highest concentration level and for preparation of further test concentrations. Presence of undissolved test item (i.e. a Tyndall effect) was not observed at the end of the stirring phase. All test concentrations were visually clear throughout the exposure intervals. Twenty daphnids were exposed to each concentration level and the control. 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. The concentrations of the test item were analytically verified via HPLC-DAD at the start (0 and 24 hours) and at the end of the exposure intervals (24 and 48 hours) in all relevant concentration levels and the control. Due to 100% mortality after 24 hours, analyses of the two highest concentration level of 20.0 and 40.0 mg/L was not carried out. The measured concentrations of the test item at the start of the exposure intervals (0 and 24 hours) were in the range of 101 to 109% of the nominal concentrations. At the end of the exposure intervals (24 and 48 hours), the measured concentrations of the test item were in the range of 98 to 106% of the nominal concentrations. The measured test item concentrations were all within ± 20% of the nominal concentrations. Indicating that the test item concentrations were successfully maintained for the duration of the exposure. Therefore, the EC50-values were based on analytically confirmed nominal concentrations of test item. The water quality parameters (i.e. pH-value and dissolved oxygen concentration), measured at the start (0 and 24 hours) and at the end of the exposure intervals (24 and 48 hours), were within the acceptable limits. The validity criteria of the test guideline were fulfilled. The 48h-EC50 was 10.1 mg/L (C.I: 5.42 – 18.90 mg/L) based on analytically confirmed nominal concentrations of the test item in the test system.

Description of key information

EC50 (invertebrates) = 3.18 (C.I. 2.91 - 3.93) mg/L ; geometric mean measured concentrations, 48hour-freshwater, OECD TG 202, 2015

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

3.18 mg/L

Additional information

Key study : OECD TG 202, 2015 : The acute toxicity to Daphnia magna was carried out according to OECD TG 202 Daphnia sp., Acute Immobilisation Test and EU Method C.2 guidelines under GLP. The study was conducted under semi-static conditions over a period of 48 hours with five concentration levels of the test item in the range of 0.50 to 8.0 mg/L prepared in a geometric series with a separation factor of 2. A stock solution of 8.0 mg/L was prepared with ISO test water (according to OECD 202, Annex 3) which was stirred, with a magnetic stirrer at approximately 1100 rpm for two hours and was used as highest concentration level and for preparation of further test concentrations. Presence of undissolved test item (i.e. a Tyndall effect) was not observed at the end of the stirring phase. All test concentrations were visually clear throughout. Twenty daphnids were exposed to each concentration level and the control. 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. The concentrations of the test item were analytically verified via GC-MS at the start (0 and 24 hours) and at the end of the exposure intervals (24 and 48 hours) in all relevant concentration levels and the control. The measured concentrations of the test item at the start of the exposure intervals (0 and 24 hours) were in the range of 64 to 102% of the nominal concentrations. At the end of the exposure intervals (24 and 48 hours), the measured concentrations of the test item were in the range of 62 to 93% of the nominal concentrations. The geometric mean measured concentrations were: 0 (control), 0.392, 0.86, 1.65, 2.79 and 6.56 mg/L. The EC50-values were based on geometric mean measured concentrations of test item. The water quality parameters (i.e. pH-value and dissolved oxygen concentration), measured at the start (0 and 24 hours) and at the end of the exposure intervals (24 and 48 hours), were within the acceptable limits. The validity criteria of the test guideline were fulfilled. The 48h-EC50 was 3.18 mg/L (C.I: 2.91 – 3.93 mg/L) based on geometric mean measured concentrations of the test item in the test system.