(1'R,2R)-2-Methyl-4-(2',2',3'-tri-nal and methylcyclopent-3'-en-1'-yl)-4-pent-enal

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:

16-08-2000 to 29-11-2000

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

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: February 2000 ; signature: April 2000

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0 (control), 0.0708, 0.132, 0.210, 0.342, 0.796, 1.41, 2.47, 4.29 and 6.05 mg/L (nominal ; based on the results of the pre-study media preparation trials and range-finder).
Time Weighted Mean Measured equivalent concentrations: 0 (control), of 0.0713, 0.116, 0.179, 0.273, 0.700, 1.29, 2.26, 3.42 and 5.32 mg/L
- Sampling method: Where possible all samples were prepared for analysis on the day of sampling. Duplicate samples were taken and stored frozen (approximately -20°C) for further analysis if necessary.
- Sample storage conditions before analysis: See above.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Direct dissolution of test item in culture medium to prepare stock; serial dilution of stock to prepare test media. The test concentrations used in the definitive study were prepared by diluting (with reconstituted water) a saturated solution prepared from an initial test item dispersion at a concentration of 1000 mg/L. Prior to addition of the test item, the reconstituted water was stirred by magnetic stirrer using a stirring rate such that a vortex depth of approximately 5% of the overall height of the water column was achieved. An amount of test item (10.0 g) was added to the vortex of 10 litres of reconstituted water to give an initial test item dispersion at a concentration of 1000 mg/L. This was stirred for 23 hours. The stirring was stopped after 23 hours and the mixture allowed to stand for 1 hour, prior to removal of the aqueous phase by siphon to give a saturated solution with a measured test concentration of 6.05 mg/L. Aliquots (50, 90, 500 and 900 ml) of this saturated solution were each separately dispersed in reconstituted water and the volume adjusted to 5 litres to provide the measured concentration of 0.0708, 0.132, 0.796 and 1.41 mg/L, whilst further aliquots (64, 112, 640 and 1120 ml) were separately dispersed in a final volume of 2 litres of reconstituted water to provide the measured concentrations of0.210, 0.342, 2.47 and 4.29 mg/L. Varying volumes of test media were prepared due to the sampling requirements of the study. The concentration and stability of the test item in the test solutions (centrifuged and uncentrifuged) were verified by chemical analysis at 0 and 48 hours. During validation: Recoveries were in the region of 98 to 111% at 0.105 to 11.1 mg/L concentrations. Based on the results of the range-finding study the test item solutions for the definitive study were prepared by stirring an excess of test item (equivalent to 1000 mg/L) in reconstituted water for a period of time and then removing the aqueous phase by siphon to produce a saturated solution with a measured concentration of 6.05 mg/L. This saturated solution was then further diluted, as necessary, to produce the remaining measured test concentrations of 0.0708, 0.132, 0.210, 0.342, 0.796, 1.41, 2.47 and 4.29 mg/L.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Not applicable.
- Concentration of vehicle in test medium (stock solution and final test solution): Not applicable.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): No precipitate reported.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain: not reported
- Source: in-house laboratory cultures
- Age at study initiation (mean and range, SD): < 24 hours
- Method of breeding: Parthenogenesis
- Feeding during test: No. The daphnids were not fed during the study. During culture: The culture daphnids are fed daily ad libitum with algae suspension
- Food type: Not applicable.
- Amount: Not applicable.
- Frequency: Not applicable.

ACCLIMATION
- Acclimation period: None reported. Can be presumed to be < 2 hours in dilution water.
- Acclimation conditions (same as test or not): Yes.
- Type and amount of food: Not applicable.
- Feeding frequency: Not applicable.
- Health during acclimation (any mortality observed): Not applicable.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Remarks on exposure duration:

In accordance with OECD TG 202 guideline.

Hardness:

Dilution water quality parameters: 0 hours: 250 mg CaCO3/L

Test temperature:

Actual: 21ºC

pH:

Control: 0 hours: pH 7.9 and 48 hours: 7.9 ; test groups: 0 hours: pH 7.9 and 48 hours: 7.9

Dissolved oxygen:

Control: 0 hours: 8.5 mg O2/L and 48 hours: 8.1 - 8.2 mg O2/L ; test groups: 0 hours: 8.4 - 8.5 mg O2/L and 48 hours: 8.1-8.2 mg O2/L

Salinity:

Not applicable.

Nominal and measured concentrations:

Definitive test: 0 (control), 0.0708, 0.132, 0.210, 0.342, 0.796, 1.41, 2.47, 4.29 and 6.05 mg/L (nominal ; based on the results of the pre-study media preparation trials and range-finder).
Time Weighted Mean Measured equivalent concentrations: 0 (control), of 0.0713, 0.116, 0.179, 0.273, 0.700, 1.29, 2.26, 3.42 and 5.32 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass jars
- Type (delete if not applicable): open, but covered to reduce test item losses
- Material, size, headspace, fill volume: glass; 250 mL fill volume, headspace ca. 0 mL (minimal)
- Aeration: No aeration of the test solutions.
- No. of organisms per vessel: Control and test item: 20 per concentration, 10 per vessel (divided into 2 replicates)
- No. of vessels per concentration (replicates): 2 (two replicates, 10 daphnia per vessel).
- No. of vessels per control (replicates): 2 (two replicates, 10 daphnia per vessel).
- No. of vessels per vehicle control (replicates): Not applicable.
- Biomass loading rate: equivalent to 25 mL test media per animal

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reconstituted water - analytical grade salts dissolved in purified water equivalent or similar to ISO Test water, according to OECD 202, Annex 3: composition. pH 7.8 (adjusted if necessary).
- Total organic carbon: Not reported
- Particulate matter: Not reported
- Metals: Not reported
- Pesticides: Not reported
- Chlorine: Not reported
- Alkalinity: Not reported
- Ca/mg ratio: Not reported
- Conductivity: <5 µS/cm
- Culture medium different from test medium: No
- Intervals of water quality measurement: 0 hours and 48 hours, (dissolved oxygen, pH and temperature)

OTHER TEST CONDITIONS
- Adjustment of pH: No.
- Photoperiod: 16 h light / 8 hours dark, 20 minute dawn and dusk transition periods.
- Light intensity: Not reported

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Immobility (or adverse effects 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.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Between 1.86 and 1.4.
- Justification for using less concentrations than requested by guideline: Not applicable.
- Range finding study: Yes.
- Test concentrations: 0 (control), 0.12, 1.2 and 12 mg/L ; nominal test item concentration.
- Results used to determine the conditions for the definitive study: Yes.

Reference substance (positive control):

no

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.4 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: (95% CL. 0.33-0.50 mg/L) ; time weighted mean measured concentrations

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

0.51 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: (95% CL. 0.42-0.64 mg/L) ; time weighted mean measured concentrations

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.179 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: time weighted 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. It was speculated the decline in measured test concentrations was therefore considered to be due to possible accumulation of the test item in the test organisms or to any organic waste matter present in the test system.
- Effect concentrations exceeding solubility of substance in test medium: No.

Reported statistics and error estimates:

The EC50 values and associated confidence limits at 24 and 48 hours were calculated by the maximum-likelihood probit method (DJ Finney, 1971) using a ToxCalc computer software package (v5.0.23C).

Table 1.0: Cumulative immobilisation data in the definitive test

Nominal Concentration (mg/L) *	Time Weighted Mean Measured Concentration (mg/L)	Cumulative population immobilised (n=20)
		24 hours				48 hours
		R1	R2	Total	%	R1	R2	Total	%
0 (control)	0	0	0	0	0	0	0	0	0
0.0708	0.0713	0	0	0	0	0	0	0	0
0.132	0.116	0	0	0	0	0	0	0	0
0.210	0.179	0	0	0	0	0	0	0	0
0.342	0.273	3	2	5	25	4	3	7	35
0.796	0.700	5	6	11	55	8	8	16	80
1.41	1.29	10	10	20	100	10	10	20	100
2.47	2.26	10	10	20	100	10	10	20	100
4.29	3.42	10	10	20	100	10	10	20	100
6.05	5.32	10	10	20	100	10	10	20	100

*Measured concentrations of the freshly prepared centrifuged test media at 0 hours

Validity criteria fulfilled:

yes

Conclusions:

The test item 48h-EC50 was 0.40 mg/L (C.I: 0.33 – 0.50 mg/L) based on time weighted 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. Following preliminary range finding test, twenty daphnids in two replicates of 10 were exposed for 48 hours under static test conditions to an aqueous solution of the test item at the following nominal concentrations:0 (control), 0.0708, 0.132, 0.210, 0.342, 0.796, 1.41, 2.47, 4.29 and 6.05 mg/L. The equivalent Time Weighted Mean Measured concentrations were: 0 (control), of 0.0713, 0.116, 0.179, 0.273, 0.700, 1.29, 2.26, 3.42 and 5.32 mg/L based on the results of chemical analysis at 0 hours, 24 hours and 48 hours.The test item solutions for the definitive study were prepared by stirring an excess of test item (equivalent to 1000 mg/L) in reconstituted water for a period of time and then removing the aqueous phase by siphon to produce a saturated solution with a measured concentration of 6.05 mg/L. This saturated solution was then further diluted, as necessary, to produce the remaining measured test concentrations. Samples of centrifuged and uncentrifuged test media were analysed to determine the amount of dissolved test item present within the test system.The number of immobilised Daphnia were recorded after 24 and 48 hours. Physicochemical measurements on water quality were made throughout the test including: temperature, pH and oxygen concentration. The temperature was maintained at 21°C and the pH was 7.9 and the dissolved oxygen concentrations were > 8.0 mgO2/L. The validity criteria of the test guideline were fulfilled. A marked decline was shown in the measured concentrations of the old or expired test media with values ranging from 0.0553 mg/L to 4.83 mg/L for uncentrifuged samples and 0.0718 mg/L to 4.66 mg/L for the centrifuged samples. On this basis the test item was not maintained at ±20% of the nominal/initial concentrations therefore the effect levels were estimated based on time weighted mean measured concentrations. Under the conditions of this study, the 48h-EC50 was 0.40 (C.I: 0.33 – 0.50) mg/L based on time weighted mean measured concentrations.

Description of key information

EC50 (invertebrates) = 0.40 (C.I: 0.33 – 0.50) mg/L based time weighted mean measured concentrations, 48hour-freshwater, OECD TG 202, 2000

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.4 mg/L

Additional information

Key study : OECD TG 202, 2000 : 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. Following preliminary range finding test, twenty daphnids in two replicates of 10 were exposed for 48 hours under static test conditions to an aqueous solution of the test item at the following nominal concentrations:0 (control), 0.0708, 0.132, 0.210, 0.342, 0.796, 1.41, 2.47, 4.29 and 6.05 mg/L. The equivalent Time Weighted Mean Measured concentrations were: 0 (control), of 0.0713, 0.116, 0.179, 0.273, 0.700, 1.29, 2.26, 3.42 and 5.32 mg/L based on the results of chemical analysis at 0 hours, 24 hours and 48 hours.The test item solutions for the definitive study were prepared by stirring an excess of test item (equivalent to 1000 mg/L) in reconstituted water for a period of time and then removing the aqueous phase by siphon to produce a saturated solution with a measured concentration of 6.05 mg/L. This saturated solution was then further diluted, as necessary, to produce the remaining measured test concentrations. Samples of centrifuged and uncentrifuged test media were analysed to determine the amount of dissolved test item present within the test system.The number of immobilised Daphnia were recorded after 24 and 48 hours. Physicochemical measurements on water quality were made throughout the test including: temperature, pH and oxygen concentration. The temperature was maintained at 21°C and the pH was 7.9 and the dissolved oxygen concentrations were > 8.0 mgO2/L. The validity criteria of the test guideline were fulfilled. A marked decline was shown in the measured concentrations of the old or expired test media with values ranging from 0.0553 mg/L to 4.83 mg/L for uncentrifuged samples and 0.0718 mg/L to 4.66 mg/L for the centrifuged samples. On this basis the test item was not maintained at ±20% of the nominal/initial concentrations therefore the effect levels were estimated based on time weighted mean measured concentrations. Under the conditions of this study, the 48h-EC50 was 0.40 (C.I: 0.33 – 0.50) mg/L based on time weighted mean measured concentrations.