Reaction products of cyclododeca-1,5,9-triene and dinitrogen monoxide, distillation overheads

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Cyclododeca-1,5,9-triene

EC Number:

225-533-8

EC Name:

Cyclododeca-1,5,9-triene

Cas Number:

4904-61-4

Molecular formula:

C12 H18

IUPAC Name:

cyclododeca-1,5,9-triene

Details on test material:

- Name of test material (as cited in study report): Cyclododeca-1,5,9-triene
- Analytical purity: no data

Specific details on test material used for the study:

name : 1,5,9-cyclododecatriene
CAS registry no. : 4904-61-4
Batch / Lot no. : A-655
Lab journal no. : 3343-13997
Date of manufacture : 02.06.2009
Expiry date : 02.06.2010
Storage conditions : RT
State of matter : liquid
Appearance / color : clear
Composition : 98 % cis, trans, trans-1,5,9-cyclododecatriene, 1.4 % trans, trans, trans-1,5,9-cyclododecatriene, 0.37 % cis, cis, trans-1,5,9-cyclododecatriene, 0.014 % cis, cis, cis-1,5,9-cyclododecatriene
Molecular formula : C12H18
Molecular weight : 162.3
Purity : 99.8%
Vapor pressure : ca. 0.1 hPa (20°C)
Density : 0.892 g/cm3
Solubility : 0.005 g/l in H20

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 100 (0.12) mg/L , nominal (measured)
- Sampling method: samples drawn from additional beakers without algae, 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

Vehicle:

no

Details on test solutions:

One 5 L graduated flask was prepared. One 2 L beaker was filled with 1.5 L of culture medium serving as control. The following test substance preparation was used: 500 mg 1,5,9-cyclododecatriene + 5000 mL culture medium. 5 L culture medium was filled into the graduated flask, stirred with a magnetic stirrer (300 rpm) and the corresponding amount of the test substance was added to the stirring medium. The flask was immediately closed by a plug and the preparation was stirred for 24h.
Afterwards the test substance preparation was filtered through a filter paper circle Ø 320 mm (Whatman Schleicher & Schuell, Dassel, Germany) into a second 5 L graduated flask that was also closed by a plug. Due to the volatile properties of the test substance care was taken that air contact was reduced to an absolute minimum, i.e. flasks and test vessels were closed immediately after handling and any unnecessary procedures such as decanting, re-filling, etc. were avoided. The control medium was treated in the same way.
For the control group, 8 test vessels (300 mL) were filled with 150 mL culture medium. One vessel was used as blank; one served for analytical determination. 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.
For the treatment group, separate test vessels were prepared per replicate for each measuring time point (0, 24, 48, 72h). In order to minimise volatilisation, each vessel was sampled only once (for photometrical measurement or analytical determinations) and was discarded after measurement. In total, 32 test vessels were filled each with 150 mL of the prepared test concentration (100 mg/L nominal). The appropriate volume (containing 7.5 x 105 cells) of the algae pre-culture was added to six replicate sets. Two additional replicate sets without algae were used as blank for the photometric measurement and for analytical determinations.
Four test vessels (see above) were prepared for the analytical determinations at 0, 24, 48 and 72 hours. 150 mL test substance preparation (100 mg /L nominal) were filled into each conical flask and were stored under the same conditions as the test vessels with algae. After 0, 24, 48 and 72 hours, the respective test vessels were sealed with parafilm and were transferred to the test site and analytical determination was 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 was transferred: 1 mL of the stock culture was diluted in two 300 mL conical flasks with 100 mL of fresh sterile culture medium. After two weeks, these stock cultures were discarded and new cultures started as described above.

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:

yes

Total exposure duration:

72 h

Test conditions

Test temperature:

24.9 - 25.2 °C

pH:

7.81 - 9.63

Nominal and measured concentrations:

100 mg/L nominal
0.12 mg/L 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
- Control end cells density: 698780 cells /mL
- No. of vessels per concentration (replicates): 3 (3 for each measuring time point 0, 24, 48, 72 h respectively)
- No. of vessels per control (replicates): 6

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 / 8 h dark
- 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: none, limit test
- Justification for using less concentrations than requested by guideline: limit test as described by guideline
- Range finding study: 0.1, 1, 10, 100 mg/L nominal
- Test concentrations: 100 mg/L nominal
- 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.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.12 mg/L

Nominal / measured:

meas. (initial)

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:
- 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 (t-test (homog. Variances)) determined after 72 hours exposure ofDesmodesmus subspicatusto the test substance 1,5,9-cyclododecatriene

 

	Effective concentrations (mg/L)
	nominal concentrations		actual concentrations
	Yield	Growth rate		Yield	Growth rate
EC10       (0-72h)	>100	>100		>0.12	>0.12
EC20       (0-72h	>100	>100		>0.12	>0.12
EC50       (0-72h)	>100	>100		>0.12	>0.12
95 % Confidence interval	n.d.	n.d.		n.d.	n.d.
LOEC    (0-72h)	>100	>100		>0.12	>0.12
NOEC    (0-72h)	≥100	≥100		≥0.12	≥0.12

 

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

 

EC values could not be determined by the statistical program ToxRat®, because no inhibition of algae growth occurred.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The ErC50 (72 h), based on measured concentrations was determined as > 0.12 mg/L (measured mean initial), which constitutes the limit of solubility of the test substance under the conditions of this test. This indicates that the test substance does not pose a significant hazard to algae.

Executive summary:

The test substance 1,5,9 -cyclododecatriene 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 algaeDesmodesmus subspicatusas 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 100 mg/L culture medium (limit test).During this study the test substance concentration of nominal 100mg/Lculture medium and the control (culture medium) was determined by the test site AQura GmbH by gas chormatography.The results of this study show that the test substance 1,5,9 -cyclododecatriene induced no inhibition of algal growth at the limit test concentration of 100 mg/L nominal (0.12 mg/L measured) with an EC50 (72h) for growth rate of > 0.12 mg/L (measured mean initial) and an NOEC for growth rate of >= 0.12 mg/L (measured mean initial), which constitutes the solubility limit of the test substance under test conditions.