3-(4,4-dimethylcyclohex-1-en-1-yl)propanal

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:

08-03-2010 to 28-02-2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

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: November 2007; signature: November 2008

Analytical monitoring:

yes

Details on sampling:

- Concentrations: The undiluted filtrate with the loading rate of 100 mg/L and dilutions of 1:3.2, 1:10, 1:32, 1:100 and 1:320 were used as test media. Additionally, a control (test water without test item) was tested in parallel.
i.e. 0 (control), 1:320, 1:100, 1:32, 1:10, 1:3.2 and undiluted filtrate loading rate 100 mg/L (based on the results of the pre-study media preparation trials and range-finder).
Equivalent geometric Mean Measured equivalent concentrations: 0 (control), not analysed, not analysed, 1.4, 4.5, 17, 56 mg/L, respectively
Note: The samples of the two lowest test concentrations (dilution 1:320 and 1:100) were not analysed since these concentrations were below the 48-hour NOEC determined in this test.
- Sampling method: For the determination of the actual test item concentrations, duplicate samples were taken from each treatment before the test start and at the end of the test after 48 hours. For the 48-hour stability samples, the contents of the respective replicates were combined prior to sampling. Immediately after sampling, acetonitrile was added to each sample in the ratio of 1:1 to stabilise the samples during the storage period. All samples were stored deep-frozen (at about -20 °C). Based on pre-experiments for investigation of the storage stability (non-GLP), the test item proved to be sufficiently stable in the test water under these storage conditions.
- 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: (non-GLP) range finding test: 0 (control), 1:100, 1:10 and 100 mg/L (loading rate/dilution rate) e.g. ca. 0 (control), 1, 10 and 100 mg/L nominal test item. 100% immobilisation was observed at 1:10 and 100 mg/L respectively. 0% at 1:100 mg/L loading rate/dilution rate.
For the definitive test: The dosing of test item was conducted as follows. Due to the low water solubility of the test item, a dispersion with the loading rate of 100 mg/L was prepared by mixing 121.7 mg of the test item into 1218 mL test water. No auxiliary solvent or emulsifier was used. The test item was mixed into the test water as homogeneously as possible using intense stirring for 3 hours at room temperature in the dark. The stirring period of 3 hours was chosen according to the results of a pre-experiment in order to maximise the test item amount in test medium. The dispersion of the test item was let to settle for 30 minutes in a separation funnel. The lower and upper phases were then removed and the middle phase was filtered through a membrane filter (0.45 micrometre pore size). The negative pressure of the filtration unit was reduced as far as possible to avoid losses of test item. The undiluted filtrate was used as the highest concentrated test medium and as stock solution for the preparation of the test media of the lower test concentrations. For the preparation of the lower concentrated test media, the filtrate was diluted with test water. The test media were prepared just before the start of the test (start of exposure). The undiluted filtrate with the loading rate of 100 mg/L and dilutions of 1:3.2, 1:10, 1:32, 1:100 and 1:320 were used as test media. Additionally, a control (test water without test item) was tested in parallel. i.e. 0 (control), 1:320, 1:100, 1:32, 1:10, 1:3.2 and undiluted filtrate loading rate 100 mg/L (based on the results of the pre-study media preparation trials and range-finder). Equivalent Geometric Mean Measured concentrations: 0 (control), not analysed, not analysed, 1.4, 4.5, 17, 56 mg/L, respectively. Note: The samples of the two lowest test concentrations (dilution 1:320 and 1:100) were not analysed since these concentrations were below the 48-hour NOEC determined in this test.
- 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: Clone 5 (from supplier)
- Source: in-house laboratory cultures (original supplier of source documented in the full study report)
- 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 three times a week 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 (ISO medium)

Test temperature:

Actual: 21ºC

pH:

Control: 0 hours: pH 7.8 and 48 hours: 7.6 ; test groups: 0 hours: pH 7.8 and 48 hours: 7.6 - 7.9

Dissolved oxygen:

Control: 0 hours: 8.6 mg O2/L and 48 hours: 7.6 mg O2/L ; test groups: 0 hours: 8.1-8.7 mg O2/L and 48 hours: 7.0-7.7 mg O2/L

Salinity:

Not applicable.

Nominal and measured concentrations:

(non-GLP) range finding test: 0 (control), 1:100, 1:10 and 100 mg/L (loading rate/dilution rate) e.g. ca. 0 (control), 1, 10 and 100 mg/L nominal test item
Definitive test: The undiluted filtrate with the loading rate of 100 mg/L and dilutions of 1:3.2, 1:10, 1:32, 1:100 and 1:320 were used as test media. Additionally, a control (test water without test item) was tested in parallel.
i.e. 0 (control), 1:320, 1:100, 1:32, 1:10, 1:3.2 and undiluted filtrate loading rate 100 mg/L (based on the results of the pre-study media preparation trials and range-finder).
Equivalent geometric Mean Measured equivalent concentrations: 0 (control), not analysed, not analysed, 1.4, 4.5, 17, 56 mg/L, respectively
Note: The samples of the two lowest test concentrations (dilution 1:320 and 1:100) were not analysed since these concentrations were below the 48-hour NOEC determined in this test.

Details on test conditions:

TEST SYSTEM
- Test vessel: 50 mL glass tubes (completely filled to ca. 50 mL test medium)
- Type (delete if not applicable): closed glass stoppers, minimum headspace to reduce test item losses
- Material, size, headspace, fill volume: glass; 50 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, 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.
- Biomass loading rate: equivalent to 10 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. 0.75 mmol/L NaHCO3 to 0.0075 mmol/L KCl in ISO water i.e. 10:1
- Pesticides: Not reported
- Chlorine: Not reported
- Alkalinity: Not reported. 0.8 mmol/L in ISO water
- Ca/mg ratio: Not reported. 0.5 mmol/L MgSO4.7H20 to 2.0 mmol/L CaCl2.2H2O in ISO water i.e. 4:1
- 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, 30 minute transition periods.
- Light intensity: 520-680 Lux

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: 3.2 ; chosen based on the results of the (non-GLP) range-finding test, which documented a relatively flat concentration-effect relationship.
- Justification for using less concentrations than requested by guideline: Not applicable.
- Range finding study: Yes.
- Test concentrations: 0 (control), 1:100, 1:10 and 100 mg/L (loading rate/dilution rate) e.g. ca. 0 (control), 1, 10 and 100 mg/L nominal test item concentration. 100% immobilisation was observed at 1:10 and 100 mg/L respectively. 0% at 1:100 mg/L loading rate/dilution rate.
- Results used to determine the conditions for the definitive study: Yes.

Reference substance (positive control):

no

Remarks:

potassium dichromate ; was reported in the last non-concurrent sensitivity check : 48-hour EC50: 0.43 mg/L (which was within the expected range of 0.43 -1.1 mg/L)

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

2.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: (95% CL. 1.4-4.5 mg/L) ; geometric mean measured concentrations

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

2.9 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: (95% CL. 2.3-3.5 mg/L) ; geometric mean measured concentrations

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

1.4 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 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 reported.
- Effect concentrations exceeding solubility of substance in test medium: No.

Reported statistics and error estimates:

The 24-hour EC50 and the 95% confidence limits were calculated by Probit Analysis.
The 48-hour EC50 could not be calculated by Probit Analysis or Moving Average Interpolation due to the steep concentration-effect relationship. Instead, the 48-hour EC50 was determined as the geometric mean value of the two consecutive test concentrations with 0 and 100% immobility (EC0 and EC100). The confidence limits for the EC50 correspond to the EC0 and EC100 values.
All reported biological results are based on mean measured test item concentrations. The lowest test concentrations (dilutions 1:320 and 1:100) were not included in the Probit Analysis calculation, as they were below the 48-hour NOEC, and thus, not analysed.

Table 1.0 : Immobilisation data in the definitive test

Treatment / Dilution	Geometric Mean Measured Concentration (mg/L)	No. Daphnids tested	Immobilised 24 hours		Immobilised 48 hours
			No.	%	No.	%
control	0	20	0	0	0	0
Dilution 1:320	n.a.	20	0	0	0	0
Dilution 1:100	n.a.	20	0	0	0	0
Dilution 1:32	1.4	20	0	0	0	0
Dilution 1:10	4.5	20	19	95	20	100
Dilution 1:3.2	17	20	20	100	20	100
Undiluted filtrate (loading rate: 100 mg/L)	56	20	20	100	20	100

n.a. = not analysed

Validity criteria fulfilled:

yes

Conclusions:

The test item 48h-EC50 was 2.5 (C.I: 1.4 – 4.5) 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. Following preliminary range finding testing, twenty daphnids in four replicates of 5 were exposed for 48 hours under static test conditions to an aqueous solution of the test item. Due to the low water solubility of the test item, a dispersion of the test item with the loading rate of 100 mg/L was prepared. The dispersion was continuously stirred at room temperature in the dark over 3 hours. After the stirring period, the dispersion was filtered through a membrane filter and the undiluted filtrate (saturated solution) was used as highest test concentration and as stock solution for the preparation of the lower concentrated test media. The following treatments were tested: Dilution 1:320, 1:100, 1:32, 1:10, 1:3.2 and the undiluted filtrate in parallel to a control. At the start of the test, the analytically measured concentrations in the dilutions 1:32, 1:10, 1:3.2 and in the undiluted filtrate (loading rate of 100 mg/L) were 1.6, 5.0, 18 and 57 mg/L, respectively. At the end of the test, the measured values decreased to 1.3, 4.2, 16 and 55 mg/L, respectively. The biological results were related to the mean measured test item concentrations calculated as the geometric means of the concentrations measured at the start and at the end of the test. The mean measured concentrations for the dilutions 1:32, 1:10, 1:3.2 and the undiluted filtrate were 1.4, 4.5, 17 and 56 mg/L, respectively. The two lowest test concentrations were not analysed since they were below the 48-hour NOEC determined in this test. Under the conditions of this study, the 48h-EC50 was 2.5 (C.I: 1.4 – 4.5) mg/L based on geometric mean measured concentrations. The NOEC was 1.4 mg/L.

Description of key information

48h-EC50 (invertebrates) = 2.5 (C.I: 1.4 – 4.50) mg/L based on geometric mean measured concentrations, 48 -hour-freshwater, OECD TG 202, 2011

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

2.5 mg/L

Additional information

Key study : OECD TG 202, 2011 : 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 testing, twenty daphnids in four replicates of 5 were exposed for 48 hours under static test conditions to an aqueous solution of the test item. Due to the low water solubility of the test item, a dispersion of the test item with the loading rate of 100 mg/L was prepared. The dispersion was continuously stirred at room temperature in the dark over 3 hours. After the stirring period, the dispersion was filtered through a membrane filter and the undiluted filtrate (saturated solution) was used as highest test concentration and as stock solution for the preparation of the lower concentrated test media. The following treatments were tested: Dilution 1:320, 1:100, 1:32, 1:10, 1:3.2 and the undiluted filtrate in parallel to a control. At the start of the test, the analytically measured concentrations in the dilutions 1:32, 1:10, 1:3.2 and in the undiluted filtrate (loading rate of 100 mg/L) were 1.6, 5.0, 18 and 57 mg/L, respectively. At the end of the test, the measured values decreased to 1.3, 4.2, 16 and 55 mg/L, respectively. The biological results were related to the mean measured test item concentrations calculated as the geometric means of the concentrations measured at the start and at the end of the test. The mean measured concentrations for the dilutions 1:32, 1:10, 1:3.2 and the undiluted filtrate were 1.4, 4.5, 17 and 56 mg/L, respectively. The two lowest test concentrations were not analysed since they were below the 48-hour NOEC determined in this test. Under the conditions of this study, the 48h-EC50 was 2.5 (C.I: 1.4 – 4.5) mg/L based on geometric mean measured concentrations. The NOEC was 1.4 mg/L.