Cyclododecanone

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-11-27 to 2009-12-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline Study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Cyclododecanone

EC Number:

212-595-6

EC Name:

Cyclododecanone

Cas Number:

830-13-7

Molecular formula:

C12H22O

IUPAC Name:

cyclododecanone

Details on test material:

- Name of test material (as cited in study report): Cyclododecanon
- Physical state: solid
- Analytical purity: > 99.5 %
- Composition of test material, percentage of components: 99.5 - 99.9 % Cyclododecanone, 0.1 - 0.3 % Zwischensieder
- Lot/batch No.: 3347-27103
- Expiration date of the lot/batch: 2010-08-18

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 6.25 (2.93), 12.5, 25 (11.96), 50, and 100 (21.91) mg/L nominal (measured)
- Sampling method: samples drawn from additional beakers without daphnia, kept under the same experimental conditions
- Sample storage conditions before analysis: samples were drawn, immediately transferred to the analytical laboratory and analysed on the same day.

Test solutions

Details on test solutions:

Six 2 L beakers were prepared. One was filled with 1.5 L of culture medium for the control. The following preparations of Cyclododecanon were used: 6.25 mg + 1000 mL culture medium; 12.5 mg + 1000 mL culture medium; 25 mg + 1000 mL culture medium; 50 mg + 1000 mL culture medium; 100 mg + 1000 mL culture medium. The volumes of culture medium were weighed into five 2 L beakers each, stirred with a magnetic stirrer (300 rpm) and the corresponding amounts of the test substance were added to the stirring medium. Preparations were stirred for 24h.
Afterwards the test substance preparations were filtered through a filter paper circle Ø 320 mm (Whatman Schleicher & Schuell, Dassel, Germany). The control was treated the same way.
For the control group, 7 test vessels (300 mL) were filled with 150 mL culture medium. One vessel was used as blank; the others were inoculated with 7.5 x 105 cells (final concentration 5 x 103 cells per mL) of algal suspension (pre-culture) according to the calculation of the concentration from the measurement with the Coulter Counter.
Four test vessels were filled each with 150 mL of the prepared test concentrations; the remaining solution was used to determine the pH-value at the beginning of the test and for analytical determinations.
Four additional samples in 300 mL conical flasks were prepared for analytical determinations at the test site AQura GmbH, Marl, Germany. 300 mL test solution from the concentration levels 6.25; 25; 100 mg test substance/L culture medium and pure culture medium (control, only 0h) were filled each into the conical flasks and stored under the same conditions as the test vessels. After 0 h and 72 h, 30 mL of each sample were transferred to a 50 mL glass flask, which were closed with screw cap and were immediately shipped to the test site and analytical determination were performed on the same day.

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Desmodesmus subspicatus
- Strain: 86.81
- Source (laboratory, culture collection): Collection of Algae Cultures, Institute for Plant Physiology, University of Göttingen, Germany
- Age of inoculum (at test initiation): < 2 weeks
- Method of cultivation: A slant agar tube with Desmodesmus subspicatus is stored in the refrigerator. Two 300 mL conical flasks, filled with 100 mL of sterile culture medium were inoculated with algae from the slant agar. Twice a week, the algae stock culture is transferred: One mL of the stock culture was diluted in two 300 mL conical flasks with 100 mL of fresh sterile culture medium.


ACCLIMATION
- Acclimation period: none
- Culturing media and conditions (same as test or not): not
- Any deformed or abnormal cells observed: no such cells observed

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

24.7 - 24.9 °C

pH:

7.81 - 8.82

Nominal and measured concentrations:

6.25 (2.93), 12.5, 25 (11.96), 50, and 100 (21.91) mg/L nominal (measured)

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: glas, 300 mL volume, filled with 150 mL culture medium
- Aeration: none
- Renewal rate of test solution (frequency/flow rate): static test
- Initial cells density: 5000 cells/mL
- No. of vessels per concentration (replicates): 3 with algal suspensions, 1 for analytical sampling
- No. of vessels per control (replicates): 6 with algal suspensions, 1 for analytical sampling
- No. of vessels per vehicle control (replicates): none


GROWTH MEDIUM
- Standard medium used: yes
- Detailed composition if non-standard medium was used: -


TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: yes


OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 16 h light /
- Light intensity and quality: lighting supplied by fluorescent tubes Osram Dulux L, “Cool White” was 0.72 x 1020 photons per m2s = 8000 +/- 1000 Lux
- Salinity (for marine algae): -


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: spectrophotometer
- Chlorophyll measurement: -

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Justification for using less concentrations than requested by guideline: -
- Range finding study
- Test concentrations: 0.1 mg/L, 1.0 mg/L, 10 mg/L, 100 mg/L
- Results used to determine the conditions for the definitive study: growth inhibition observed in pre-test

Reference substance (positive control):

yes

Remarks:

potassium dichromate was tested in an independent experiment in October 2009 and adequate sensitivity of the algal culture was demonstrated

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

7.77 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

4.63 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test):
- Unusual cell shape: no
- Colour differences: no
- Flocculation: no
- Adherence to test vessels: no
- Aggregation of algal cells: no
- Other: no
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50: 0.8 mg/L
- Other:

Reported statistics and error estimates:

Statistical analysis was done with ToxRat Professional (ToxRat Solutions GmbH, Alsfeld, Germany)

Any other information on results incl. tables

Table: EC (probit analysis using linear max. likelihood regression), LOEC and NOEC values (Williams Multiple Sequential t-test for Yield/Welch-t-test for Inhomogeneous Variances with Bonferroni Adjustment for Growth rate) determined after 72 hours exposure of Desmodesmus subspicatus to different concentrations of test substance Cyclododecanon

 

	Effective concentrations (mg/L)
	nominal concentrations		actual concentrations
	Yield	Growth rate		Yield	Growth rate
EC10       (0-72h)	9.18	11.88		5.56	4.63
EC20       (0-72h	10.38	13.65		6.12	5.53
EC50       (0-72h)	13.13	17.79		7.34	7.77
95 % Confidence interval	12.98 – 13.31	16.89 – 18.74		n.d.	n.d.
LOEC    (0-72h)	12.50	12.50		n.d.	n.d.
NOEC    (0-72h)	6.25	6.25		n.d.	n.d.

 

n.d.: not determined due to mathematical reasons or inappropriate data

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The ErC50 (72 h), based on measured concentrations was determined as 7.77 mg/L , indicating that the test substance may be toxic to aquatic invertebrates. The corresponding EC10 = 4.63 mg/L.

Executive summary:

The test substance Cyclododecanol was tested in the Alga, Growth Inhibition Test according to the OECD Guideline for the Testing of Chemicals No. 201 (23 March 2006) using the single cell green algae Desmodesmus subspicatus as test organism. The experiment was carried out as a static test, i.e. the test substance preparations were not exchanged for the whole exposure period of 72 hours. Based on non-GLP pre-test results the main test was performed with test substance concentrations of nominal 0, 6.25, 12.5, 25, 50, and 100 mg/L culture medium. During this study the test substance concentrations of nominal 6.25, 25, and 100 mg/L culture medium and the control (culture medium) were determined by the test site AQura GmbH by gas chormatography.The results of this study show that the test substance Cyclododecanon induced inhibition of algal growth with an EC50 (72h) for growth rate of 7.77 mg/L (geometric mean measured) and an EC10 (72 h) for growth rate of 4.63 mg/L (geometric mean measured).