Reaction mass of (E)-3-methylcyclotetradec-5-en-1-one and (Z)-3-methylcyclotetradec-5-en-1-one

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:

04 February to 01 April 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

conducted under GLP conditions

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

1984

Deviations:

yes

Remarks:

In the range-finding test after 24 hours of exposure, one vessel in the 0.01 mg/I group was found to contain 6 organisms instead of 5. The study integrity was not adversely affected by this deviation.

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

1992

Deviations:

yes

Remarks:

In the range-finding test after 24 hours of exposure, one vessel in the 0.01 mg/I group was found to contain 6 organisms instead of 5. The study integrity was not adversely affected by this deviation.

GLP compliance:

yes

Specific details on test material used for the study:

Batch TQT0300497
Purity 90.9%
Test substance storage At room temperature in the dark
Retest date 28 November 2004

Analytical monitoring:

yes

Details on sampling:

Sampling frequency: at t=0 h and t=48 h
Sampling volume: 1.5 ml (0.32, 0.56 and 1.0 mg/l) or 15 ml (controls, 0.10 and 0.18 mg/l) from the approximate centre of the test vessel
All samples were analysed freshly.

Vehicle:

yes

Remarks:

acetone

Details on test solutions:

The standard test procedures required generation of test solutions, which should contain completely dissolved test substance concentrations or stable and homogeneous mixtures or dispersions. The testing of concentrations that disturb the test system should be prevented (e.g. film of the test substance on the water surface).

The batch of KARMALONE tested was a colourless to pale yellow liquid with a purity of 90.9% and completely soluble in test medium at all but the highest concentration tested. Since the test substance was known to be relatively photosensitive, preparation of test solutions was performed under dimmed light conditions.

In the range-finding test, preparation of test solutions started with a stock solution of 100 mg/ml in acetone (Lab-scan), which was serially diluted in acetone to reach concentrations a factor of 10,000 above the target concentrations. After addition of 100 µI of the respective stock to ISO medium, the solutions were treated with ultrasonic waves for 5 minutes and magnetically stirred for 15 minutes. The resulting solutions were clear and colourless, except for the highest concentration, which contained undissolved particles. After 3 minutes of stabilization, the water phase was separated from this solution and used as such.

In the final test, preparation was similar as in the range-finding test, but the highest tested concentration was lower, and all test solutions were clear and colourless without stabilization.

Note that the solvent-control in the range-finding test was prepared by addition of 100 µI acetone to 1 liter of test medium without further treatment, while it was treated the same way as the test solutions in the final test.

Test organisms (species):

Daphnia magna

Details on test organisms:

Species: Daphnia magna (Crustacea, Cladocera) (Straus, 1820), at least third generation, obtained by acyclical parthenogenesis under specified breeding conditions.

Source: In-house laboratory culture with a known history

Reason for selection: This system has been selected as an internationally accepted invertebrate species.

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.

Characteristics: For the test selection of young daphnia 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, as prescribed by Dr. Elendt-Schneider (Elendt, B.-P., 1990: Selenium deficiency in Crustacea. An ultrastructural approach to antenna! damage in Oaphnia magna Straus. Protoplasma 154,
25-33).

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test temperature:

The temperature of the test medium was 19.6°C at the start of the test. The temperature continuously measured in a temperature control vessel varied between 19.9 and 20.4°C during the test, and complied with the requirements as laid down in the protocol (18-22°C, constant within 2°C).

pH:

between 7.7 and 8.1

Dissolved oxygen:

between 6.8 and 9.0 mg/l

Nominal and measured concentrations:

Definitive test: Nominal concentrations: 0.1, 0.18, 0.32, 0.56 and 1.0 mg/I and controls: Test medium without test substance or other additives (blank-control), and test medium without test substance but with the additive used in the treatment of the stock solutions (solvent-control).
Measured concentrations: measured concentrations were 88-106% of nominal ones. During the exposure period the measured concentration remained constant,with actual concentrations at the end of the test being 80-92% of nominal. Results were therefore based on nominal concentrations.

Details on test conditions:

Test duration: 48 hours
Test type: Static
Test vessels: 50 ml, all-glass, closed with glass stoppers
Medium: ISO
Number of daphnia: 20 per concentration
Loading: 5 per vessel containing 50 ml medium
Light: No light, the test was performed in darkness (0-1 lux)
Feeding: No feeding
Aeration: No aeration of the test solutions
Introduction of daphnia: Within 1 hour after preparation of the test solutions

Reference substance (positive control):

no

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.58 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CI 0.53-0.68 mg/L

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

Analysis of the samples taken at the start (t=0) of the final test showed that meas-ured concentrations were 88-106% of nominal. During the exposure period the measured concentration remained constant, with actual concentrations at the end of the test being 80-92% of nominal. Results were therefore based on nominal concentrations.

The immobility observed during the final test was: 0% at 0.1, 0.18 and 0.32 mg/L; 35% at 0.56mg/L; and 100% at 1mg/L. The responses recorded in this test allowed for reliable determination of an EC50. The responses recorded at 0.10 and 1.0 mg/L were in agreement with the results of the range-finding test.

The results of measurement of pH and oxygen concentrations (mg/I) are presented in Table 4 of the study report. These test conditions remained within the limits prescribed by the protocol (pH: 6.0-8.5, not varying by more than 1 unit; oxygen: >7 mg/I at the start, 25 mg/I at the end of the test).
The temperature of the test medium was 19.6°C at the start of the test. The temperature continuously measured in a temperature control vessel varied between 19.9 and 20.4°C during the test, and complied with the requirements as laid down in the protocol (18-22°C, constant within 2°C).

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the present study KARMALONE did not induce acute immobilisation of Daphnia magna at 0.32 mg/I after 48 hours of exposure (NOEC).

The 48h-EC50 was 0.58 mg/I based on nominal concentrations (95% confidence interval between 0.53 and 0.68 mg/I).

Executive summary:

The study procedures described in this report were based on the ISO International Standard 6341, 1996. In addition, the procedures were designed to meet the test methods and validity criteria of the EEC directive 92/69, Part C.2, 1992 and the OECD guideline No. 202 Part I, 1984.

The batch of KARMALONE tested was a colourless to pale yellow liquid with a purity of 90.9% and completely soluble in test medium at all but the highest concentration tested. Since the test substance was known to be relatively photosensitive, preparation of test solutions was performed under dimmed light conditions and testing was performed in darkness.

Preparation of test solutions started with a stock solution of 100 mg/ml in acetone (Lab-scan), which was serially diluted in acetone to reach concentrations a factor of 10,000 above the target concentrations. After addition of 100µI of the respective stock to ISO medium, the solutions were treated with ultrasonic waves for 5 minutes and magnetically stirred for 15 minutes. The resulting solutions were clear and colourless, except for the highest concentration in the range­ finding test, which contained undissolved particles on the bottom of the vessel. After 3 minutes of stabilization, the water phase was taken from this solution and used as such.

A range-finding test was performed exposing ten daphnia per concentration (in duplicate, 5 per vessel) to a range of 0.001 to 10 mg/L increasing by a factor of 10 and to a solvent-control and a blank-control. After 48 hours, all daphnids exposed to 1.0 and 10 mg/L were immobilized, while no immobilization occurred at 0.10 mg/L and below. Analysis of samples taken at the start and the end of the test showed that the concentration at 1.0 mg/L was stable throughout the test period.

In the final test, twenty daphnia per concentration (in quadruplicate, 5 per vessel) were exposed to 0.10, 0.18, 0.32, 0.56 and 1.0 mg/L and to a blank-control and a solvent-control. The total test period was 48 hours. Samples for analysis were taken at the start and the end of the test.Analysis of the samples showed that all test concentrations were in agreement with nominal throughout the test period.

The test met the acceptability criteria described in the protocol and was considered valid.

KARMALONE did not induce acute immobilisation of Daphnia magna at 0.32 mg/L after 48 hours of exposure (NOEC).

The 48h-EC50was 0.58 mg/L based on nominal concentrations (95% confidence interval between 0.53 and 0.68 mg/L).

Description of key information

The acute toxicity of Cosmone (Karmalone) to Daphnia magna was carried out according to OECD 202 Daphnia sp. Static Test and EU Method C.2 guidelines under GLP.

EC50(48hrs) = 0.58 mg/L (95% confident interval between 0.53 and 0.68 mg/L)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.58 mg/L

Additional information

According to the EU CLP regulation (No 1272/2008 and its adaption 286/2011), EC50(48hrs) crustacea < 1mg/L corresponds to Hazardous to the Aquatic Environment Acute 1 classification.

In the absence of chronic toxicity data for this trophic level, the long-term aquatic hazard is classified as Chronic 1 based on the EC50 of < 1mg/L and a logKow of => 4.