4-Penten-2-ol, 3,3-dimethyl-5-(2,2,3-trimethyl-3-cyclopenten-1-yl)-, (4E)-

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:

From 18 to 20 July 2012.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

This study was performed according to OECD Guideline 202 and EU Method C.2 with GLP compliance. All validity criteria were fulfilled and no deviations were observed.

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

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected on September 14, 2010 / signed on February 07, 2011

Specific details on test material used for the study:

Storage condition of test material: ambient temperature in the dark, away from heat.

Analytical monitoring:

yes

Details on sampling:

Samples of all test media were taken from the preparation flasks prior to distribution to test vessels at the test start. At test end (48 h), further samples of test media (all concentrations and control) were taken from pooled replicate vessels according to treatment. Duplicate samples of 20 mL were taken.
Analytical sub-samples of 1000 µL of the aqueous test medium were mixed together with 250 µL methanol directly into 1.8mL screw top vials. After tightly closing and vigorous manual shaking, 20 µL of the mixture was analysed directly by LC-MS/MS. Where necessary, sample aliquots less than 1000 µL were filled up to 1000 µL with 'holding- and dilution water' in a pre-dilution step.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution was prepared by weighing 20 mg of the test substance onto a Teflon plate that was then put into 2 L of dilution water. The substance was dissolved by stirring at 100 rpm for 24 hours as stated in the study plan. This formed the stock solution and highest test concentration. The remaining test concentrations were then prepared by adding aliquots of 280, 155, 85, and 50 mL (in order from highest to lowest concentration) to dilution water at a final volume of 500 mL.
- Eluate: dilution water
- Controls: Yes. The control consisted of dilution water only.
- 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 data

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain: no data
- Source: German Federal Environment Agency, Institut für Wasser-, Boden- und Lufthygiene. Specimens
- Age at study initiation (mean and range, SD): juvenile, aged less than 24 hours at the start of the test
- Weight at study initiation (mean and range, SD): no data
- Length at study initiation (length definition, mean, range and SD): no data
- Valve height at study initiation, for shell deposition study (mean and range, SD): no data
- Peripheral shell growth removed prior to test initiation: no data
- Method of breeding: bred in the laboratory at Fraunhofer IME.
Adult Daphnia, at least 3 weeks old, were separated from the stock population by sieving. Batches of 30 - 50 animals were held at room temperature in ca. 1.8 L of dilution water for one week. During this week, the daphnids were fed daily with an algal suspension (Desmodesmus subspicatus) and LiquizellR (HOBBY). Algae growing in the log-phase were centrifuged and the pellet was re-suspended in a few mL of medium. Thirty millilitres of this suspension was added to 1 L of medium. The water was changed once per week. Newborn Daphnia (between 4 - 24 h old) were removed by wide-bore pipette (to avoid damage) and isolated in fresh dilution water for at least 1 h prior to being added randomly to the test vessels containing the appropriate test or control media.
- Feeding during test: no

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Remarks on exposure duration:

none

Post exposure observation period:

None

Hardness:

1.1 mmol/L

Test temperature:

21.0°C at test start and 20.9°C at the end of the test.

pH:

The pH was within the range of 7.73 – 8.44 in the control and test media.

Dissolved oxygen:

The oxygen saturation was between 7.4 – 8.2 mg/L (91 – 99% saturation) in the control and test media.

Salinity:

Not applicable

Nominal and measured concentrations:

- Nominal concentrations: loadings of 10.0, 5.6, 3.1, 1.7, and 1.0 mg/L (spaced factor of 1.8).
- Measured concentrations: The chemical analysis of the fresh test medium at test start revealed a recovery of between 61 – 65% of the nominal. Concentrations of the aged test medium at test end were recovered at 42 – 46% of the nominal loadings (66 – 74% of the initial concentration at test start). As the concentrations decreased from the nominal loadings by more than 20%, the geometric mean of the measured concentrations of the test substance was calculated and used for reporting toxicity effects: 5.278, 2.989, 1.677, 0.895 and 0.551 mg/L. See table 6.1.3/1 in "Any other information on materials and methods incl. tables".

Details on test conditions:

TEST SYSTEM
- Test vessel: 60 mL glass beakers, covered with a glass pane
- Material, size, headspace, fill volume: Approximately 50 mL of test medium was added to each replicate test vessel
- Aeration: The test vessels were not aerated during the test.
- Renewal rate of test solution (frequency/flow rate): The test solutions were not exchanged.
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): not applicable

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Purified local tap water was used in accordance with the OECD guideline. The tap water is sourced from the Schmallenberg district water production plants, mostly fed by small springs and percolation. The purification process occurs on site at Fraunhofer IME and includes filtration with activated charcoal, passage through a lime-stone column, and aeration to the point of oxygen saturation. To reduce copper contamination (as copper is toxic to aquatic organisms), plastic water pipes are used in the test facilities.

OTHER TEST CONDITIONS
- Adjustment of pH: no data
- Photoperiod: light/dark cycle of 16/8 hours.
- Light intensity: 795 – 813 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : After 24 h and 48 h, the number of immobile animals in each beaker was counted. The animals were considered to be immobile if they were not able to swim within 15 seconds of gentle agitation of the test vessels. Any abnormalities in appearance and behaviour were also recorded.

TEST CONCENTRATIONS
- Range finding study: yes (loadings of 10, 1, 0.1 and 0.01 mg/L and a control).
- Results used to determine the conditions for the definitive study: After 48 hours, 100% immobility was observed in the highest concentration. No immobile animals were observed in the other treatments.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

1.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CL could not be determined

Duration:

48 h

Dose descriptor:

other: The highest concentration without observed effect

Effect conc.:

0.9 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- The effect evaluation was based on the geometric mean measured concentration of test substance.
- No signs of disease or stress were observed. All surviving animals gave the impression of being healthy.
- At 24 h, the three highest geometric mean measured concentrations had significantly affected immobilisation by 30, 100, and 100% compared to the control, respectively. After 48 h, the immobilisation in the 1.68 mg/L concentration had increased to 65%. Immobilisation was 5% in the control.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
The latest 24h-EC50 was 1.03 mg/L (April 2012).

Reported statistics and error estimates:

The computer software ToxRat® professional was used for the statistical evaluations.

Table 6.1.3/2: Raw data of immobility counts at 0, 24 and 48h

Geometric mean conc. [µg/L]	Control	551	895	1677	2989	5278
0 h	5	5	5	5	5	5
	5	5	5	5	5	5
	5	5	5	5	5	5
	5	5	5	5	5	5
Number of replicates:	4	4	4	4	4	4
Total Introduced:	20	20	20	20	20	20
24 h	0	0	0	2	5	5
	0	0	0	0	5	5
	0	0	0	3	5	5
	0	0	0	1	5	5
Number of replicates:	4	4	4	4	4	4
Total Immobile::	0	0	0	6	20	20
48 h	0	1*	0	3	5	5
	1	0	0	3	5	5
	0	0	0	4	5	5
	0	0	0	3	5	5
Number of replicates:	4	4	4	4	4	4
Total Immobile::	1	1	0	13	20	20

*This immobilisation effect was not considered biologically significant or a dose response. As such it was excluded from the analysis.

Table 6.1.3/3: Summary of immobilisation effects on Daphnia magna

Geometric mean conc. [mg/L]	0-24 h % immobilisation	0-48 h % immobilisation
Control	0 -	5 -
0.60	0 -	5* -
0.90	0 -	0 -
1.68	30 +	65 +
2.99	100 +	100 +
5.28	100 +	100 +

*This immobilisation effect was not considered biologically significant or a dose response. As such it was excluded from the analysis.

Validity criteria fulfilled:

yes

Remarks:

Immobilisation in the control treatment was 5% and did not exceed 10%. The dissolved oxygen concentration at the end of the aging period (48 h) was >= 3 mg/L and >60% in the control and test vessels.

Conclusions:

The EC50 for the immobilisation of Daphnia magna by the test substance was 1.60 mg/L.

Executive summary:

At the Fraunhofer Institute for Molecular Biology and Applied Ecology, the influence of the test substance on immobilisation of Daphnia magna was investigated, according to OECD Guideline 202 and EU Method C.2 with GLP statement. Daphnia were exposed under static conditions to the test item for 48 hours.

 

Based on the results of a pre-test, five nominal loadings and a control were tested: 1.0, 1.7, 3.1, 5.6, and 10.0 mg/L. The test solutions were prepared by directly dissolving the test substance off of a Teflon plate in dilution water by stirring for 24 hours. The test substance was measured in the freshly prepared test solutions at test start and in the aged test media after 48 h at test end (LOQ 100 µg/L).

 

Measured concentrations of the test substance at test start were within the range of 61.5 – 65.0% of the nominal loadings. By test end (48 h), the measured recovery range of all test concentrations was 42.9 – 47.4% of the nominal loadings (66 – 74% of the initial concentration at test start). As the recovery of the test substance was outside the range of 80 - 120 % of the nominal values, the geometric mean for each test concentration was calculated and used for the evaluation of the acute immobilisation test. The geometric mean measured concentrations were 0.60, 0.90, 1.68, 2.99, and 5.28 mg/L, respectively.

 

Effects on immobilisation were determined after 24 and 48 hours. After 48 hours, the percent immobilisation was 0, 0, 65, 100 and 100% at the respective concentrations of 0.60, 0.90, 1.68, 2.99 and 5.28 mg/L. The EC₅₀was 1.60 mg/L. The highest concentration without observed effect after 48 hours was 0.90 mg/L.

Description of key information

OECD 202, EU Method C.2, GLP, key study, validity 1:

48h-EC50 (Daphnia magna) = 1.6 mg/L based on geometric mean measured concentrations.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

1.6 mg/L

Additional information

One key study is available to assess the short-term toxicity of the registered substance to aquatic invertebrates, Daphnia magna. This study was performed according to OECD Guideline 202 and EU Method C.2 with GLP compliance under static conditions for 48 hours.

Based on the results of a pre-test, five nominal loadings of the registered substance and a control were tested: 1.0, 1.7, 3.1, 5.6, and 10.0 mg/L. The test substance was measured in the freshly prepared test solutions at test start and in the aged test media after 48 h at test end (LOQ 100 µg/L). Measured concentrations of the test substance at test start were within the range of 61.5 – 65.0% of the nominal loadings. By test end (48 h), the measured recovery range of all test concentrations was 42.9 – 47.4% of the nominal loadings (66 – 74% of the initial concentration at test start). As the recovery of the test substance was outside the range of 80 - 120 % of the nominal values, the geometric mean for each test concentration was calculated and used for the evaluation of the acute immobilisation test. The geometric mean measured concentrations were 0.60, 0.90, 1.68, 2.99, and 5.28 mg/L, respectively. Effects on immobilisation were determined after 24 and 48 hours. After 48 hours, the percent immobilisation was 0, 0, 65, 100 and 100% at the respective concentrations of 0.60, 0.90, 1.68, 2.99 and 5.28 mg/L. Therefore, the 48 hours EC₅₀was 1.60 mg/L (95% CL could not be determined) and the highest concentration without observed effect after 48 hours was 0.90 mg/L, based on geometric mean measured concentrations.