Cyclopropanecarboxylic acid, 2-[1-(3,3-dimethylcyclohexyl)ethoxy]-2-methylpropyl ester

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:

24-05-2004 to 23-06-2004

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

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: May 2002 ; signature: September 2002

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Definitive test: nominal concentrations: 0 (control), 0.5, 0.8, 1.3, 2.0 and 3.2 mg/L nominal and average exposure concentrations of the three highest test concentrations: 0.97, 1.48, and 1.49 mg/L, respectively; controls: Test medium without test substance or other additives. Spacing factor determined from range-finding test and physico-chemical properties of the substance.
- Sampling method: Singular samples for possible analysis were taken from all test concentrations and the control according to the schedule:
1. At the start of the test 0-hours from the freshly prepared solutions.
2. The end of the test from the 48-hour old solutions, volume: 4.5 mL for controls, 0.5, 0.8 and 1.3 mg/L test item groups; 1.5 mL for 2.0 and 3.2 mg/L test item groups ; from the approximate centre of the test vessels.
- Sample storage conditions before analysis: Samples were used on day of sampling; reserve samples were stored in a freezer until analysis (≤ -15°C). Reserve samples of 15 mL were taken for possible analysis if needed.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: For the purpose of the definitive test, the test item was dispersed in acetone due to limited water solubility. Preparation of test solutions started with stock solutions in acetone at a factor of 10,000 higher than the final test solutions. Volumes of 100 μL were then added per litre ISO-medium to obtain the desired concentration. The highest concentration prepared during the range-finding test was then magnetically stirred for 40 minutes, while the two lower concentrations were stirred for 10 minutes after which the respective solutions were used for testing. Preparation during the final test included a magnetic stirring period of 30 minutes, which was followed by a stabilisation period of 20-30 minutes, except for the two lowest test concentrations that were fully dissolved. The water phase was collected in case a test substance film was observed after the stabilisation period. The final test solutions were all clear and colourless except the highest concentration tested in the range-finding test, which contained a thin floating layer. Test solutions were renewed after 24 hours during the final test.
- Eluate: Not applicable.
- Differential loading: Not applicable.
- Controls: Solvent control was used (containing the dispersant only)
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Acetone
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): See above.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): No.
- Other relevant information: Not applicable.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphinds
- Strain: Daphnia magna (Crustacea, Cladocera) (Straus, 1820), at least third generation, obtained by acyclical parthenogenesis under specified breeding conditions.
Source: in-house laboratory cultures
- Age at study initiation (mean and range, SD): < 24 hours.
- Method of breeding: Parthenogenesis
- Feeding during test: No.
- Food type: Not applicable
- Amount: Not applicable.
- Frequency: Not applicable.

ACCLIMATION
- Acclimation period: None.
- Acclimation conditions (same as test or not): Not applicable.
- Type and amount of food: Not applicable.
- Feeding frequency: Not applicable.
- Health during acclimation (any mortality observed): Not applicable.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Remarks on exposure duration:

In accordance with the OECD TG 202 guidelines.

Hardness:

The ISO medium hardness: 250 mg/L expressed as CaCO3 and the pH: 7.9 ± 0.2

Test temperature:

The temperature continuously measured in a temperature control vessel varied between 20.4 and 20.9°C during the test, and complied with the requirements as laid down in the protocol (18-22°C, constant within 2°C).

pH:

Test conditions remained within the limits prescribed by the guideline; pH: 7.8-8.0, not varying by more than 1.5 unit at the end of the test.

Dissolved oxygen:

Test conditions remained within the limits prescribed by the guideline; oxygen: >= 3 mg/l at the end of the test.

Nominal and measured concentrations:

Range-finding test: 0 (control), 0.05, 0.5, and 5.0 mg/L nominal; plus Control containing no test substance.
Definitive test: nominal concentrations: 0 (control), 0.5, 0.8, 1.3, 2.0 and 3.2 mg/L nominal and average exposure concentrations of the three highest test concentrations: 0.97, 1.48, and 1.49 mg/L, respectively; controls: Test medium without test substance.

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL all glass closed
- Type (delete if not applicable): closed ; semi-static
- Material, size, headspace, fill volume: Glass
- Aeration: No aeration of the test solutions.
- No. of organisms per vessel: Control and test item: 20 per concentration, 5 per vessel containing 50 mL of test solution
- 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: Reverse Osmosis (RO-water, GEON Waterbehandeling, The Netherlands); used to prepare ISO Medium M7
- Culture medium different from test medium: No, ISO Medium M7, renewed by 50% twice per week.

OTHER TEST CONDITIONS
- Adjustment of pH: None.
- Photoperiod: 16 hours light; 8 hours dark photoperiod daily
- Light intensity: Not reported.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Immobility (including mortality), 24 hours and at 48 hours.

RANGE-FINDING STUDY
- Test concentrations: 0 (control), 0.05, 0.5, and 5.0 mg/L nominal
- 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.:

> 1.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CL: - mg/L

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 1.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CL: - mg/L

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.97 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other:

Remarks:

The NOEC was set at 0.97 mg/L despite the fact that one immobile daphnia was observed. A maximum response of 10% immobilisation is acceptable for the control and the absence of a clear treatment related response.

Details on results:

- Behavioural abnormalities: None
- Observations on body length and weight: Not applicable.
- Other biological observations: None.
- Mortality of control: No mortalities.
- Other adverse effects control: None.
- Abnormal responses: None.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No specific comments made. Analysis of the samples taken at the start of the final test showed measured concentrations of 1.2, 1.7 and 1.8 mg/L at the nominal concentrations of 1.3, 2.0 and 3.2 mg/L, respectively. Measured concentrations in the freshly prepared solutions after 24 hours were comparable to those measured at the start, thus showing that preparation was repeatable. The measured concentrations remained generally stable within 20% during the 24-hour renewal periods, except for the highest concentration measured at the end of the test (77% of initial}. Measured concentrations at nominally 2.0 and 3.2 mg/L differed only marginally from each other, which indicated that the maximum soluble concentration was to be expected just below 2.0 mg/L. Based on the results of analysis the maximum solubility in test medium was approximately 1.5 mg/L.
- 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 0.84 mg/L. 48h-EC50 was 0.661 mg/L with a 95% confidence interval between 0.54 and 0.87 mg/L
- Other: The actual responses in this reference test with K2Cr2O7 are within the ranges of the expected responses at the different concentrations, i.e. the 48h-EC50 was between 0.3 and 1.0 mg/L. Hence, the sensitivity of this batch of test organism was in agreement with the historical data..

Table 1. pH and oxygen (mg/L) concentrations during the definitive test

	pH values				Dissolved oxygen concentration (mg/L)
Nominal Concentration (mg/L)	Initial 0 hours	t=24h old	t=24h fresh	End 48 hours	Initial 0 hours	t=24h old	t=24h fresh	End 48 hours
0 (Control)	7.8	7.9	7.9	7.9	9.0	9.4	9.6	8.8
0.5	7.8	7.8	7.9	7.9	9.1	9.4	9.6	8.9
0.8	7.8	7.8	7.8	8.0	9.1	9.3	9.6	8.9
1.3	7.8	7.8	7.8	8.0	9.1	9.3	9.6	8.9
2.0	7.8	7.8	7.8	8.0	9.1	9.3	9.6	8.9
3.2	7.9	7.8	7.8	8.0	9.1	9.3	9.6	8.9

 

Table 2. Number of introduced daphnids and incidence of immobility in the definitive test

Nominal concentration (mg test item/L)	Replicate	Number of daphnids exposed	Response at 24h		Response at 48h
			Number	Total %	Number	Total %
Solvent Control	1 2 3 4	5 5 5 5	0 0 0 0	0	0 0 0 0	0
0.5	1 2 3 4	5 5 5 5	0 0 0 0	0	0 0 0 0	0
0.8	1 2 3 4	5 5 5 5	0 0 0 0	0	0 0 0 0	0
1.3	1 2 3 4	5 5 5 5	0 0 0 0	0	0 0 1 0	5
2.0	1 2 3 4	5 5 5 5	0 (1) 0 0 0 (1)	0	1 1 1 (1) 0	15
3.2	1 2 3 4	5 5 5 5	0 (2) 0 (3) 1 (4) 2 (4)	15	1 1 1 2	25

brackets: number of daphnia trapped at the surface. These organisms were re-immersed into the respective solutions before recording of mobility.

Validity criteria fulfilled:

yes

Conclusions:

The test item 48h-EC50 was > 1.5 mg/L (C.I: – mg/L), which corresponds to the maximum solubility of the test item, based on geometric mean measured average exposure concentrations that were analytically confirmed.

Executive summary:

The acute toxicity to Daphnia magna was carried out according to OECD 202 Daphnia sp., Acute Immobilisation Test and EU Method C.2 guidelines under GLP. Following preliminary range finding test in the 0.5 to 5.0 mg/L nominal range using acetone dispersant to increase water solubility, no significant immobilisation was observed at 0.05 and 0.5 mg/L. A total of 90% of the daphnia became immobilised at 5.0 mg/L. lt should however be recognised that daphnia in the highest concentration were exposed to an over saturated solution as an oily test substance floating layer was present in which the immobilised daphnia were trapped. The EC50 was expected to be above the limit of solubility based on the observations during the range-finding test. An identically conducted definitive study was completed in 100 mL closed glass flasks under semi-static conditions. Preparation of test solutions started with stock solutions in acetone at a factor of 10,000 higher than the final test solutions. Volumes of 100 μL were then added per litre ISO-medium to obtain the desired concentration. The highest concentration prepared during the range-finding test was then magnetically stirred for 40 minutes, while the two lower concentrations were stirred for 10 minutes after which the respective solutions were used for testing. Preparation during the final test included a magnetic stirring period of 30 minutes, which was followed by a stabilisation period of 20-30 minutes, except for the two lowest test concentrations that were fully dissolved. The water phase was collected in case a test substance film was observed after the stabilisation period. The final test solutions were all clear and colourless except the highest concentration tested in the range-finding test, which contained a thin floating layer. Test solutions were renewed after 24 hours during the final test. The definitive test concentrations were 0 (control), 0.5, 0.8, 1.3, 2.0 and 3.2 mg/L nominal and average exposure concentrations of the three highest test concentrations: 0.97, 1.48, and 9.3 mg/L, respectively. Twenty daphnids per group (5 per replicate, quadruplicate) were exposed to an untreated control at the specified nominal test substance concentrations. All test solutions were renewed after 24 hours and samples for analytical confirmation of actual exposure concentrations were taken from freshly prepared solutions at the start and after 24 hours of exposure and from 24-hour old solutions after 24 and 48 hours of exposure. Analysis of the samples taken during the final test from the three highest concentrations showed that preparation was repeatable. Measured concentrations generally remained stable within 20% during the 24-hour renewal periods, except for the highest concentration measured at the end of the test. Based on the results of analysis the maximum solubility in test medium was approximately 1.5 mg/L. The study met the acceptability criteria prescribed by the protocol and was considered valid. The test item did not induce biologically significant immobilisation of Daphnia magna at a nominal concentration of 1.3 mg/L, corresponding to an average exposure concentration of 0.97 mg/L after 48 hours of exposure (NOEC). Significant immobilisation was observed at the two highest test concentrations: 15% at nominal 2.0mg/L (average exposure concentration of 1.48mg/L) and 25% at nominal 3.2mg/L (average exposure concentrations of 1.49 mg/L). The measured concentrations at nominally 2.0 and 3.2mg/L differed only marginally from each other, which indicated that the maximum solubility in test medium was approximately 1.5 mg/L. The 48h-EC50 was above the solubility limit of the test item in the test medium, i.e. above an average exposure concentration of 1.5 mg/L. Hence, due to the relatively low water solubility of the test substance, a 50% effect concentration could not be reached in the soluble range of the test item

Description of key information

EC50-48h (invertebrates) : > 1.5 mg/L (95% CL: – mg/L) based on geometric mean measured average exposure concentrations, 48-hour, freshwater, OECD 202, 2004

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

1.5 mg/L

Additional information

Key data: OECD TG 202, 2004 : The acute toxicity to Daphnia magna was carried out according to OECD 202 Daphnia sp., Acute Immobilisation Test and EU Method C.2 guidelines under GLP. Following preliminary range finding test in the 0.5 to 5.0 mg/L nominal range using acetone dispersant to increase water solubility, no significant immobilisation was observed at 0.05 and 0.5 mg/L. A total of 90% of the daphnia became immobilised at 5.0 mg/L. lt should however be recognised that daphnia in the highest concentration were exposed to an over saturated solution as an oily test substance floating layer was present in which the immobilised daphnia were trapped. The EC50 was expected to be above the limit of solubility based on the observations during the range-finding test. An identically conducted definitive study was completed in 100 mL closed glass flasks under semi-static conditions. Preparation of test solutions started with stock solutions in acetone at a factor of 10,000 higher than the final test solutions. Volumes of 100 μL were then added per litre ISO-medium to obtain the desired concentration. The highest concentration prepared during the range-finding test was then magnetically stirred for 40 minutes, while the two lower concentrations were stirred for 10 minutes after which the respective solutions were used for testing. Preparation during the final test included a magnetic stirring period of 30 minutes, which was followed by a stabilisation period of 20-30 minutes, except for the two lowest test concentrations that were fully dissolved. The water phase was collected in case a test substance film was observed after the stabilisation period. The final test solutions were all clear and colourless except the highest concentration tested in the range-finding test, which contained a thin floating layer. Test solutions were renewed after 24 hours during the final test. The definitive test concentrations were 0 (control), 0.5, 0.8, 1.3, 2.0 and 3.2 mg/L nominal and average exposure concentrations of the three highest test concentrations: 0.97, 1.48, and 9.3 mg/L, respectively. Twenty daphnids per group (5 per replicate, quadruplicate) were exposed to an untreated control at the specified nominal test substance concentrations. All test solutions were renewed after 24 hours and samples for analytical confirmation of actual exposure concentrations were taken from freshly prepared solutions at the start and after 24 hours of exposure and from 24-hour old solutions after 24 and 48 hours of exposure. Analysis of the samples taken during the final test from the three highest concentrations showed that preparation was repeatable. Measured concentrations generally remained stable within 20% during the 24-hour renewal periods, except for the highest concentration measured at the end of the test. Based on the results of analysis the maximum solubility in test medium was approximately 1.5 mg/L. The study met the acceptability criteria prescribed by the protocol and was considered valid. The test item did not induce biologically significant immobilisation of Daphnia magna at a nominal concentration of 1.3 mg/L, corresponding to an average exposure concentration of 0.97 mg/L after 48 hours of exposure (NOEC). Significant immobilisation was observed at the two highest test concentrations: 15% at nominal 2.0mg/L (average exposure concentration of 1.48mg/L) and 25% at nominal 3.2mg/L (average exposure concentrations of 1.49 mg/L). The measured concentrations at nominally 2.0 and 3.2mg/L differed only marginally from each other, which indicated that the maximum solubility in test medium was approximately 1.5 mg/L. The 48h-EC50 was above the solubility limit of the test item in the test medium, i.e. above an average exposure concentration of 1.5 mg/L. Hence, due to the relatively low water solubility of the test substance, a 50% effect concentration could not be reached in the soluble range of the test item