Cyclopropanemethanol, 1-methyl-2-[(1,2,2-trimethylbicyclo[3.1.0]hex-3-yl)methyl]-

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March to July 1998

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

- Name of test material (as cited in study report): Javanol
- Physical state: Pale yellow viscous liquid
- Analytical purity: 88.4% (GC, sum of two peaks)
- Lot/batch No.: 008825

Analytical monitoring:

yes

Remarks:

GC-FID

Details on sampling:

Stock culture: algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at 23 +/- 2°C.
Pre-culture: 4 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 20000 cells/ml. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.

Vehicle:

yes

Remarks:

A stock solution of JAVANOL was prepared at 100 mg/mL in acetone. A 1 in 10'000-dilution was performed with ISO Medium and stirred for 24 hours followed by filtration through a 0.2 um filter.

Details on test solutions:

For the final test, a stock solution in acetone was prepared at 100 mg/ml and diluted to 10 mg/l in ISO medium. This solution was stirred for 24h followed by filtration through a 0.2 micrometer filter. The test solutions 10, 18, 32, 56% of filtrate were prepared by subsequent dilutions..
Due to high inhibition at the highest concentrations, a second test was performed.
The same procedure for preparation was followed as in the first final test except that the dilutions prepared were 1.0, 2.2, 4.6, 10, 22 and 46% of filtrate. All were tested, including the undiluted stock (100%).

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

This system is an unicellulal algal species sensitive to toxic substances in the aquatic ecosystem and has been selected as an internationallly accepted species.

Test type:

static

Water media type:

freshwater

Remarks:

ISO Medium, according to ISO Standard "Algal Growth inhibition test" November, 1989.

Limit test:

no

Total exposure duration:

72 h

Test temperature:

Between 21.0 and 23.0°C

pH:

Between 8.0 and 8.5

Nominal and measured concentrations:

Samples were analysed at Time 0, 24 hours and 72 hours. The time.weighted average exposure concentrations were calculated for the four highest test concentrations (100%, 46%, 22% and 10% dilution of stock solution). In the 4.6%, 2.2% and 1.0% dilution of the stock exposure solutions, no effects were observed on the algae.

Nominal Solution Concn. (%) TWA Concn. (mg/L)
10% 0.14
22% 0.31
46% 0.84
100% 2.07

No detects were made in the Solvent Control.

Details on test conditions:

Test type: Static
Test vessels: 100 ml, all-glass
Milli-Q water: Tap wate purified by reverse osmosis and then passed over activated carbon and ion-exchange cartridges
Cell density: An initial cell density of 10000 cells/ml.
Test duration: 72h
Illumination: Continuously using TLD lamps of 18 watt yiedling 7500 - 8000 lux in the first EC50 test and 6500-7500 lux in the final test
Incubation: During incubation the algal cells were kept in suspension by continuous shaking

Reference substance (positive control):

yes

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.33 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% CI : 0.13 to 0.88 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.74 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI : 0.32 to 1.7 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.14 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: No Observed Effect Concentration for Biomass and Growth Rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.15 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% CI : 0.05 to 0.41 mg/L.

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.18 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI : 0.08 to 0.40 mg/L

Details on results:

Results based on time-weighted average exposure concentrations and reported in Amendment No.1 to Final Report.

Results with reference substance (positive control):

The 72h EC50 for growth inhibition was 1.3 mg/l with a 95% confidence interval of 0.34 - 4.6 mg/l.
The 72h EC50 fro growth rate reduction was 1.7 mg/l with a 95% confidence interval of 1.1 - 2.8 mg/l.
These results were in agreement with what was expected on the basis of historical data:
The historical ranges of the 72h EC50 for growth inhibition lie between 0.32 and 1.8 mg/l and the 72h EC50 for growth rate reduction lie between 0.56 and 3.2 mg/l.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the present study with Selenastrum capricornutum, Javanol inhibited cell growth and reduced growth rate on this fresh water algae species significantly at average measured concentrations of 0.31 mg/l and higher.
The EC50 for cell growth inhibition (EC50:0-72h) was 0.33 mg/l with a 95% confidence interval ranging from 0.13 to 0.88 mg/l. The EC10 for cell growth inhibition (EC10:0-72h) was 0.15 mg/l with a 95% confidence interval ranging from 0.05 to 0.41 mg/l.
The EC50 for growth rate reduction was 0.74 mg/l with a 95% confidence interval ranging from 0.32 to 1.7 mg/l. The EC10 for growth rate reduction was 0.18 with a 95% confidence interval ranging from 0.08 to 0.40 mg/l.
The NOEC corresponded with an average measured concentration of 0.14 mg/l.

Executive summary:

The study procedures described in this report were based on the EEC directive 92/69, publication no L383 part C-3 adopted December 1992; OECD guideline No. 201, Adopted june 7, 1984; and ISO standard 8692, first edition, 15 November 1989.

Javanol is a clear viscous liquid which is hardly soluble in water. The water solubility of Javanol is 3.8 mg/l (at 20°C) according to data of the sponsor. The procedure for preparation was based on research by the Analytical departement during method development and validation. This procedure included filtration of the stock solution through a 0.2 micrometer filter. Before filtration the test medium was exposed for one day to the test substance under continuous stirring to ensure maximum saturation.

After a range-finding test, two full tests were performed. The test solutions contained various amounts of a 0.2 micrometer filtrate prepared at nominal 10 mg/l with acetone as presolvent (0.1 ml/l). Analytical support showed that the algal suspensions were exposed to a range of initial Javanol concentrations of 0.4 to 4 mg/l in the first test and of 0.02 to 2 mg/l in the second and final test. The initial cell density was 10^4 cells/ml. The total test period was 72 hours.

Analysis of the samples taken at the end of the first full test showed that the actual test concentrations could not be maintained at 80% of the initial concentration, including the concentration in the solution without algae (76%). Instability increased with decreasing concentration. However, the analytical results of the second study showed that actual concentrations remained at 80% of initial in samples taken from the highest concentration without algae and in reserve samples taken from the lower concentrations. Hence EC50 values could be based on average concentrations.

Javanol inhibited cell growth and reduced growth rate of this fresh water algae species significantly at average measured concentrations of 0.31 mg/l and higher.

The EC50 for cell growth inhibition (EC50:0-72h) was 0.33 mg/l with a 95% confidence interval ranging from 0.13 to 0.88 mg/l. The EC10 for cell growth inhibition (EC10:0-72h) was 0.15 mg/l with a 95% confidence interval ranging from 0.05 to 0.41 mg/l.

The EC50 for growth rate reduction was 0.74 mg/l with a 95% confidence interval ranging from 0.32 to 1.7 mg/l. The EC10 for growth rate reduction was 0.18 with a 95% confidence interval ranging from 0.08 to 0.40 mg/l.

The NOEC corresponded with an average measured concentration of 0.14 mg/l.

Description of key information

Under the conditions of the present study with Selenastrum capricornutum, Javanol inhibited cell growth and reduced growth rate on this fresh water algae species significantly at average measured concentrations of 0.31 mg/L and higher.

The EC50 for cell growth inhibition (EC50:0-72h) was 0.33 mg/L with a 95% confidence interval ranging from 0.13 to 0.88 mg/L. The EC10 for cell growth inhibition (EC10:0-72h) was 0.15 mg/L with a 95% confidence interval ranging from 0.05 to 0.41 mg/L.

The EC50 for growth rate reduction was 0.74 mg/L with a 95% confidence interval ranging from 0.32 to 1.7 mg/L. The EC10 for growth rate reduction was 0.18 with a 95% confidence interval ranging from 0.08 to 0.40 mg/L.

The NOEC corresponded with an average measured concentration of 0.14 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

0.74 mg/L

EC10 or NOEC for freshwater algae:

0.18 mg/L

Additional information

Under the conditions of the present study with Selenastrum capricornutum, Javanol inhibited cell growth and reduced growth rate on this fresh water algae species significantly at average measured concentrations of 0.31 mg/L and higher.

The EC50 for cell growth inhibition (EC50:0-72h) was 0.33 mg/L with a 95% confidence interval ranging from 0.13 to 0.88 mg/L. The EC10 for cell growth inhibition (EC10:0-72h) was 0.15 mg/L with a 95% confidence interval ranging from 0.05 to 0.41 mg/L.

The EC50 for growth rate reduction was 0.74 mg/L with a 95% confidence interval ranging from 0.32 to 1.7 mg/L. The EC10 for growth rate reduction was 0.18 with a 95% confidence interval ranging from 0.08 to 0.40 mg/L.

The NOEC corresponded with an average measured concentration of 0.14 mg/L.