2-(3,5-dimethylhex-3-en-2-yloxy)-2-methylpropyl cyclopropanecarboxylate

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From 25 February to 05 May 2009

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Limit test, spiked with solvent at a nominal test concentration of 14 mg/L (maximum water solubility), but only 2 mg/L was detected as exposure concentration. No EC50 could be calculated because the test substance proved to be non-toxic at this concentration.

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1010 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

not specified

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

not specified

Qualifier:

equivalent or similar to guideline

Guideline:

ISO 6341 (Water quality - Determination of the Inhibition of the Mobility of Daphnia magna Straus (Cladocera, Crustacea))

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Storage conditions: At room temperature in the dark
- Stability under storage conditions: Stable
- Solubility in water: 14 mg/L

Batch No. : LDP0800001
Purity : 97.0%
Date of Expiry : 01 May, 2010

Analytical monitoring:

yes

Remarks:

GC-FID

Details on sampling:

- Sampling method: Samples for possible analysis were taken from both the limit concentration and the control at t=0h and t=48h. Volume = 15 mL from the approximate centre of the test vessels. Additionally, reserve samples of 15 mL were taken for possible analysis. If not already used, these samples were stored in a freezer for a maximum of three months.
- Sample storage conditions before analysis: Samples were stored in a freezer until analysis.

Vehicle:

yes

Remarks:

DMSO at a concentration of 0.1 mL/L.

Details on test solutions:

- Preparation of the test solution: Preparation of test solutions started with stock solutions in DMSO of 1.4, 14 and 140 mg/mL (range finding test) or 140 mg/mL only (limit test). Subsequently, 0.1 mL of the stock solution was added per litre of test medium in order to reach the test concentrations. After a 15-minute stirring period, the clear and colourless solutions were siphoned into the test vessels.
Control: Test medium without test substance but with DMSO in the same amount as used in the treatment of the stock solutions.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna (Crustacea, Cladocera)
- Strain/clone: Strain 1820. At least third generation, obtained by acyclical parthenogenesis under specific breeding conditions.
- Validity of batch: Daphnids originated from a healthy stock, 2nd to 5th brood, showing no signs of stress such as mortality > 20%, presence of males, ephippia or discoloured animals and there was no delay in the production of the first brood.
- Age at study initiation (mean and range, SD): For the test selection of young daphnids with an age of <24 hours, from parental daphnids of more than two weeks old.

BREEDING
- Start of each batch: With newborn daphnids, i.e. less than 3 days old, by placing about 250 of them into 5 litres of medium in an all-glass culture vessel.
- Maximum age of the cultures: 4 weeks
- Renewal of the cultures: After 7 days of cultivation half of the medium twice a week.
- Temperature of medium: 18-22°C
- Feeding: Daily, a suspension of fresh water algae.
- Medium: M7

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Hardness:

143 mg/L expressed as CaCO3

Test temperature:

The temperature of the test mediu was 20.2°C at the start of the test. The temperature continuously measured in a temperature control vessel varied between 19.8 and 20.7°C during the test, and complied with the requirements as laid down in the protocol (18-22°C, constant within 2°C).

pH:

From 7.8 to 8.1, so within the limits prescribed by the protocol (pH: 6.0-8.5, not varying by more than 1.5 unit)

Dissolved oxygen:

From >8 to 9.2 mg/L, so within the limits prescribed by the protocol (oxygen >= 3 mg/L at the end of the test)

Salinity:

not applicable

Conductivity:

no data

Nominal and measured concentrations:

Nominal test concentration: 14 mg/L.
Analysis of the sample taken from the limit concentration at the start of the test showed a measured concentration of 2.0 mg/L. During the exposure period, the measured concentration remained constant (i.e. 106% of initial). Therefore, effect parameters were based on the initial exposure concentration. See table 6.1.3/1 in "Any other information on results incl. tables"

Details on test conditions:

TEST SYSTEM
- Test vessel:
100 mL, all glass, closed with a screw cap which was additionally sealed with laboratory film (parafilm). Small nets were used in the vessels to prevent the daphnids from getting trapped at the surface
- Type (delete if not applicable): closed
- Aeration:
no aeration of the test solutions
- No. of organisms per vessel:
5
- No. of vessels per concentration (replicates):
4
- No. of vessels per vehicle control (replicates):
4
- Biomass loading rate:
5 per vessel containing 80 mL of test solution
- Introduction of daphnids: immediately after preparation of the test solutions.

TEST MEDIUM / WATER PARAMETERS
Adjusted ISO medium with a hardness of 143 mg/L CaCO3

OTHER TEST CONDITIONS
- Photoperiod:
16 hours photoperiod daily

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Immobility (including mortality): at 24 hours and at 48 hours
- pH and dissolved oxygen: At the beginning and at the end of the test, for both the limit concentration and the control
- Temperature of medium: continuously in a temperature control vessel, beginning at the start of the test

TEST CONCENTRATIONS
- Preliminary data: A non-GLP pretest was performed by Givaudan. This experiment shows that a solution of the test substance in ISO medium does not remain stable (65.8-70.3% of initial) without agitation at least for 96 hours in closed glass containers (full or not). When stored in an open vessel, the substance had evaporated out of the solution almost totally after 96 hours (1.4% initial).
- Range-finding study: A range-finding test was performed to provide information about the range of concentrations to be used in the final test. Test procedure and conditions were similar to those applied in the final limit test with the following exceptions: Ten daphnids per concentration (in duplicate, 5 per vessel) were exposed to 0.14, 1.4 and 14 mg test substance/L and to a control; Dissolved oxygen concentrations and pH were only measured in the control and the highest test concentration; No sampling for determination of actual test concentrations was performed; No nets were used in the test vessels.
After 48h of exposure, 20% immobility was observed at nominal 14 mg/L. However, from 24h onwards all daphnids exposed to this concentration were observed to be trapped at the surface. No floating layer could be seen but it was expected that this concentration was slightly above the solubility limit of the test substance in test medium. No immobility was observed at any of the lower test concentrations and the control during the test period. The expected EC50 was above nominal 14 mg/L. All test conditions were maintained within the limits prescribed by the protocol. For the limit test, it was decided to use nets in the test vessels to prevent daphnids from getting trapped at the surface.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

>= 2 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: Observable effects

Remarks on result:

other: No effects observed on daphnids up to and including the highest test concentration, considered to represent the saturation concentration, 2 mg/L.

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 2 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

No immobility nor other effects were observed in both the limit concentration and the control. No daphnids became trapped at the surface during the test. See table 6.1.3/2 in "Any other information on results incl. tables".
No EC50 could be calculated because the test substance proved to be non-toxic (EC50 > maximum soluble concentration tested).

Results with reference substance (positive control):

- Results with reference substance valid? yes
The actual responses in this reference test with potassium dichromate were within the ranges of the expected responses. However, the sensitivity of this batch of Daphnia magna was slightly higher when compared with the historical data collected at NOTOX. The 24h-EC50 and 48h-EC50 values were 0.55 mg/L (95% CI: 0.50 - 0.63 mg/L) and 0.30 mg/L (95% CI: 0.25 - 0.35 mg/L), respectively.

Table 6.1.3/1: Concentrations of the test substance in test medium (limit test)

Time of sampling (hours)	Date of sampling (dd-mm-yy)	Date of analysis* (dd-mm-yy)	Concentration
			Nominal (mg/L)	Analysed (mg/L)	Relative to nominal (%)	Relative to initial (%)
0	17-03-09	23-03-09	0 14	n.d. 2.01	n.a. 14
48	19-03-09	23-03-09	0 14	n.d. 2.14	n.a. 15	106

* Samples were stored in the freezer until the day of analysis

n.d.: not detected

n.a.: not applicable

Table 6.1.3/2: Acute immobilisation of daphnids after 24 and 48 hours in the limit test

Concentration of test substance (mg/L)	Vessel number	Number daphnia exposed	Response at 24h		Response at 48h
			Number	Total %	Number	Total %
Control	A B C D	5 5 5 5	0 0 0 0	0	0 0 0 0	0
2.0	A B C D	5 5 5 5	0 0 0 0	0	0 0 0 0	0

Validity criteria fulfilled:

yes

Remarks:

In the control, no daphnids became immobilised; The oxygen concentration at the end of the test was >=3 mg/L in control and test vessels.

Conclusions:

Under the conditions of the present study, the test substance did not induce acute immobilisation or any other observable effects on Daphnia magna at 2.0 mg/L after 48 hours of exposure. The 48h-EC50 was above 2 mg/L, which was considered to be the maximum solubility of the test substance in ISO-medium. The NOEC is > = 2 mg/L.

Executive summary:

This study was performed to assess the acute toxicity of the test substance in Daphnia magna according to the EPA Guideline OPPTS 850.1010 with GLP compliance. In addition, the procedures were designed to meet the test methods of the OECD 202, EU Method C2 and ISO 6341 Guidelines..

Based on the results of a range-finding test, a limit test was performed. Preparation of test solutions started with a stock solution in DMSO of 140 mg/mL. Subsequently, 0.1 mL of the stock solution was added per litre of test medium in order to reach the nominal test concentration of 14 mg/L. After a 15-minute stirring period, the clear and colourless solution was siphoned into the test vessels. Twenty daphnids per concentration (four replicates, five daphnids per replicate) were exposed to a control group and the nominal test concentration of 14 mg/L. Test vessels were closed and the total test period was 48 hours. Samples for analytical confirmation of actual exposure concentrations were taken at the start and the end of the test.

The study met acceptability criteria prescribed by the protocol and was considered valid.

Analysis of the sample taken from the limit concentration at the start of the test showed a measured concentration of 2.0 mg/L. During the exposure period, the measured concentration remained constant (e.i. 106% of initial). Therefore, effect parameters were based on the initial exposure concentration.

The test substance did not induce acute immobilisation nor any other observable effects on Daphnia magna at 2.0 mg/L after 48 hours of exposure. The 48h-EC50 was above 2 mg/L, which was considered to be the maximum solubility of the test substance in ISO-medium.  The NOEC is > = 2 mg/L.

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 22 September 2020 to 17 December 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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 12 June 2019 / Date of signature: 26 August 2019

Specific details on test material used for the study:

- Solubility in water: 14 mg/L at 20°C
- Vapour pressure: 0.0046 hPa at 20°C (OECD 104)
- Conditions of storage: In a closed container in a dry, well ventilated dark location at ambient temperature/10–30°C

Batch No. : SC00029369
Purity : 97.6%
Date of Expiry : 19 May, 2022

Analytical monitoring:

yes

Remarks:

GC-MS

Details on sampling:

- Concentrations: Samples of stock solution and test solutions were taken to determine the actual test item concentrations.
- Sampling method: Control and test solution were sampled in duplicate. The duplicate samples were kept separately as a reserve. The samples of freshly prepared test and control solutions were taken directly from the volumetric flasks in which they had been prepared. The samples of 24-hour-aged test and control solutions were taken from the respective test vessels. A mixing of test solutions from replicates was not performed prior to sampling. All sampling steps were performed using glass equipment.
- Sample storage conditions before analysis: After sampling and before shipment, all samples were stored in glass bottles in the dark at a temperature of ≤ -18 °C. A non-GLP storage stability test was performed at the analytical test site prior to the conduct of the definitive biological phase, which confirmed sample integrity through frozen storage.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: See details in "Any other information on materials and methods, incl. tables".
- Medium: Test item dissolved in medium M4

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna (Straus)
- Strain/clone: clone M10
- Age at study initiation (mean and range, SD): Less than 24 hours
- Age of parental stock (mean and range, SD): Less than 35 days
- Holding conditions / Food and feeding frequency: Algae (Desmodesmus subspicatus), instant baker’s yeast suspension, TetraMin® suspension; 3–4 times per week
- Feeding during test: none

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

239.3 mg CaCO3/L

Test temperature:

From 19.5 to 21.2°C

pH:

From 7.7 to 7.8

Dissolved oxygen:

From 98 to 103% of air saturation, corresponding to 8.5-9.5 mg O2/L

Salinity:

not applicable

Conductivity:

662 µS/cm

Nominal and measured concentrations:

- Nominal loading rates: 100, 50.0, 25.0, 12.5 and 6.25% of a saturated solution at a loading rate of 100 mg/L.
- Measured concentrations: See table 6.1.3/6 in "Any other information on results incl. tables". Geometric mean measured concentrations = 0.349 - 0.730 - 1.33 - 3.24 and 6.04 mg test item/L

Details on test conditions:

TEST SYSTEM
- Test vessel: glass vials (nominal volume 20-mL) air-tightly closed by a screw-cap with PTFE-lined septum
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 25 mL (vessels filled to the brim)
- Renewal rate of test solution (frequency/flow rate): renewal after 24 hours of exposure
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
Medium M4 (OECD 2004)

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light : 8 hours dark
- Light intensity: 722-756 lx

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Immobility

RANGE-FINDING STUDY
- Test concentrations: 0.8 - 4.0 - 20 - 100% of a saturated solution at a loading rate of 100 mg/L.
- Results used to determine the conditions for the definitive study: Based on the results of a preliminary non-GLP range finding test which did not show any immobility at 0.8, 4.0, 20 and 100% of a saturated solution at a loading rate of 100 mg/L, the following concentrations in a geometrical series (spacing factor: 2.0) were tested in the definitive test: 100, 50.0, 25.0, 12.5 and 6.25% of a saturated solution at a loading rate of 100 mg/L. Additionally, daphnids were exposed under control conditions (non-treated test medium).

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

>= 6.04 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Observable effects

Remarks on result:

other: No effects observed on daphnids up to and including the highest tested concentration (highest attainable test concentration).

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 6.04 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: The highest attainable test concentration

Details on results:

See Tables in "Any other information on results incl. tables".
- Biological results: No effects were observed on the daphnids up to and including the highest tested exposure concentration at 100% of a saturated solution at a loading rate of 100 mg test item/L. No immobile daphnids were recorded in the controls, at 6.25, 12.5 and 25.0% of a saturated solution at a loading rate of 100 mg test item/L. Single immobile daphnids (5% immobility) were observed at 50 and 100% of a saturated solution at a loading rate of 100 mg test item/L. These immobile daphnids were within biological variability, i.e. within the permitted validity criteria of the test, and not statistically significant (p≤0.05).
- Analytical results: The test item concentrations can be considered stable over the test period.

Results with reference substance (positive control):

The health of the Daphnia culture and the overall quality of the test organisms is monitored regularly by using the reference toxicant potassium dichromate (K2Cr2O7) in an acute test design in order to determine the sensitivity of the organisms from the culture and to demonstrate that the test conditions are appropriate.
The most recent reference test with potassium dichromate performed in a separate study (Study No. IDA2005; May 2020) resulted in an EC50(24 h) of 1.571 mg/L. According to the OECD guideline No. 202 (OECD, April 2004) EC50(24 h)-values for potassium dichromate obtained from different laboratories should range between 0.6 mg/L and 2.1 mg/L. The toxicity of the reference item is within this range. Therefore, the results of this reference test are acceptable and the test conditions are reliable and the sensitivity of the test system could be demonstrated.

Table 6.1.3/5: Cumulative number of immobile Daphnia magna after 24 and 48 hours

Nominal concentration	Total number of introduced daphnids	Total number of immobile daphnids	Immobile daphnids (% of introduced)	Total number of immobile daphnids	Immobile daphnids (% of introduced)
(% of saturated solution*)		24h	24h	48h	48h
control 6.25 12.5 25.0 50.0 100	20 20 20 20 20 20	0 0 0 0 0 0	0.0 0.0 0.0 0.0 0.0 0.0	0 0 0 0 1 1	0.0 0.0 0.0 0.0 5.0 5.0

*% of saturated solution at a loading rate of 100 mg test item/L.

 

Table 6.1.3/6: Summary of measured concentrations during the test

Nominal concentration (%*)	Test period (h)	Age of test matrix (h)	Measured concentration (mg test item/L)	Geometric mean measured concentration (mg test item/L)
0 (control) 6.25 12.5 25.0 50.0 100.0	0 0 0 0 0 0	0 0 0 0 0 0	<LOQ 0.291 0.737 0.967 2.66 5.26
6.25 12.5 25.0 50.0 100	24 24 24 24 24	24 24 24 24 24	0.279 0.338 1.05 2.37 5.06
6.25 12.5 25.0 50.0 100	24 24 24 24 24	0 0 0 0 0	0.402 1.13 1.96 4.69 6.81
0 (control) 6.25 12.5 25.0 50.0 100	48 48 48 48 48 48	24 24 24 24 24 24	<LOQ 0.456 1.01 1.55 3.75 7.35	n.a. 0.349 0.730 1.33 3.24 6.04

*% of saturated solution at a loading rate of 100 mg test item/L;

n.a. = not applicable

Validity criteria fulfilled:

yes

Remarks:

Dissolved oxygen concentration measured in test solutions and control at the end of the test >=8.5 mg/L; immobility in the controls = 0%; daphnids in the control showing signs of disease or stress = 0%

Conclusions:

The test substance did not induce acute immobilisation of Daphnia magna up to the highest attainable test concentration of 6.04 mg/L after 48 hours of exposure. The 48h-EC50 was above 6.04 mg/L, based on geometric mean measured concentrations, which was considered to be the maximum solubility of the test substance in M4-medium. No other effects were observed on the daphnids : NOEC > = 6.04 mg/L.

Executive summary:

This study was performed to assess the acute toxicity of the test substance in Daphnia magna according to OECD Guideline 202 and GLP compliance.

Based on the results of a preliminary non-GLP range finding test which did not show any immobility at 0.8, 4.0, 20 and 100% of a saturated solution at a loading rate of 100 mg/L, the following concentrations in a geometrical series (spacing factor: 2.0) were tested in the definitive test: 100, 50.0, 25.0, 12.5 and 6.25% of a saturated solution at a loading rate of 100 mg/L. The test period (exposure of test organisms to the test solutions in a semi-static system) was 48 hours. Additionally, daphnids were exposed under control conditions (non-treated test medium), for the same period of time. Four replicates, with five daphnids per replicate, were used per test item concentration and for the control.

The study met acceptability criteria prescribed by the protocol and was considered valid.

Samples of the test solutions were analysed to determine the actual levels of the test item at start and at end of each of the renewal periods during the test. Analysis of the test item in the samples was performed using a GC-MS method. The limit of quantification (LOQ) was 0.15 mg/L. The limit of detection (LOD) was defined as 30% of the LOQ (i.e. 0.045 mg/L). The test item concentration was stable during the test period. The geometric mean measured concentrations over 48 hours were determined to be: 0.349, 0.730, 1.33, 3.24 and 6.04 mg test item/L.

No effects were observed on the daphnids up to and including the highest tested exposure concentration at 100% of a saturated solution at a loading rate of 100 mg test item/L. No immobile daphnids were recorded in the controls, at 6.25, 12.5 and 25.0% of a saturated solution at a loading rate of 100 mg test item/L. Single immobile daphnids (5%) were observed at 50 and 100% of a saturated solution at a loading rate of 100 mg test item/L. These immobile daphnids were within biological variability, i.e. within the permitted validity criteria of the test, and not statistically significant (p≤0.05).

In conclusion, the test substance did not induce acute immobilisation of Daphnia magna up to the highest attainable test concentration of 6.04 mg/L after 48 hours of exposure. The 48h-EC50 was above 6.04 mg/L, based on geometric mean measured concentrations, which was considered to be the maximum solubility of the test substance in M4-medium.  No other effects were observed on the daphnids : NOEC > = 6.04 mg/L.

Description of key information

OECD Guideline 202, GLP, key study, validity 1:

48h-EC50 (Daphnia magna) > 6.04 mg/L (the highest attainable test concentration), based on geometric mean measured concentrations.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC50

Effect concentration:

> 6.04 mg/L

Additional information

To assess the short-term toxicity of the registered substance to aquatic invertebrates, two experimental studies are available.

 

The more recent study (ECT, 2020), determined as the key study, was performed according to OECD Guideline No. 202 with GLP compliance to assess the acute toxicity of the registered substance to the aquatic invertebrate Daphnia magna. Following a preliminary range-finding test, twenty daphnids per concentration (four replicates, five daphnids per replicate) were exposed to a control group and to 100, 50.0, 25.0, 12.5 and 6.25% of a saturated solution at a loading rate of 100 mg test substance/L, for a period of 48 hours under semi-static and closed test conditions. Samples of the test solutions were analysed, by GC-MS method, to determine the actual levels of the test item at start and at end of each of the renewal periods during the test. The test item concentration was stable during the test period. The geometric mean measured concentrations over 48 hours were determined at: 0.349, 0.730, 1.33, 3.24 and 6.04 mg test item/L. No effects were observed on the daphnids up to and including the highest tested exposure concentration. No immobile daphnids were recorded in the controls and at the geometric mean measured concentrations of 0.349, 0.730 and 1.33 mg/L. Single immobile daphnids (5%) were observed at the geometric mean measured concentrations of 3.24 and 6.04 mg/L. These immobile daphnids were within biological variability and not statistically significant. In conclusion, the test substance did not induce acute immobilisation of Daphnia magna up to the highest attainable test concentration of 6.04 mg/L after 48 hours of exposure. The 48h-EC50 value was determined to be above 6.04 mg/L, based on geometric mean measured concentrations, which was considered to be the maximum solubility of the test substance in M4-medium, under the test conditions.

 

The other study (NOTOX, 2009), determined as a supporting study, was performed according to the EPA Guideline OPPTS 850.1010 with GLP compliance to assess the acute toxicity of the test substance on Daphnia magna. Based on the results of a range-finding test, a limit test was performed with a nominal test concentration of 14 mg/L (prepared with DMSO as solvent). Twenty daphnids per concentration (four replicates, five daphnids per replicate) were exposed to a control group and the nominal test concentration of 14 mg/L for a period of 48 hours under static and closed test conditions. Samples for analytical confirmation of actual exposure concentrations were taken at the start and the end of the test. Analysis of the sample taken from the limit concentration at the start of the test showed a measured concentration of 2.0 mg/L. During the exposure period, the measured concentration remained constant (i.e. 106% of initial). Therefore, effect parameters were based on the initial exposure concentration. The test substance did not induce acute immobilisation of Daphnia magna at 2.0 mg/L after 48 hours of exposure. The 48h-EC50 was above 2 mg/L, which was considered to be the maximum solubility of the test substance in ISO-medium, under the test conditions.