4,4'-(9H-fluoren-9-ylidene)bis(2-chloroaniline)

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:

04 Jun - 22 Jun 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Also according to GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

adopted 2006

GLP compliance:

yes (incl. QA statement)

Remarks:

2005-11-21

Analytical monitoring:

yes

Details on sampling:

- Concentrations: The test material solution was analysed at 0 and 72 h.
- Sampling method: Solvent control replicate samples 1 - 6 were pooled. Samples from the test group replicates 1 - 3 and 4 - 6 were pooled.

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: An amount of test material (100 mg) was dissolved in dimethylformamide and the volume adjusted to 10 mL to give a 10 mg/mL solvent stock solution from which a dilution was made to give a 1 mg/mL solvent stock solution. An aliquot (300 µL) of the 1 mg/mL solvent stock solution was dispersed in 3 L of culture medium to give an initial 0.1 mg/L test material dispersion. This dispersion was stirred for 24 h using a magnetic stirrer and then filtrated (0.2 µm) to remove the undissolved test material and give a nominal stock solution of 0.004 mg/L. This final concentration value was based on the results of the pre-study media preparation trial. Stability analyses indicated that the test material was unstable in light. Therefore, the 0.1 mg/L test material dispersion was shielded from light during the 24 h stirring period.
- Controls: A vehicle control was included in the test.
- Chemical name of vehicle: Dimethylformamide
- Concentration of vehicle in test medium: 0.01%

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Strain: CCAP 276/20
- Source: Culture Collection of Algae and Protozoa (CCAP), Dunstaffnage Marine Laboratory, Oban, Argyll, UK.
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were maintained in the laboratory under constant aeration and constant illumination at 21 ± 1 °C

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

24 ± 1 °C (constant)

pH:

test start: 7.3 - 7.4
test end: 7.9 - 8.1

Nominal and measured concentrations:

Nominal: control, 0.004 mg/L
Measured: < LOQ, 0.00107 mg/L (geom. mean)

Details on test conditions:

TEST SYSTEM
- Test vessel: Conical flasks
- Type (delete if not applicable): closed (polyurethane foam bungs)
- Material, size, fill volume: Glass, 250 mL, 100 mL
- Aeration: Shaking at 150 rpm
- Initial cells density: 3.86E03 cells/mL
- Control end cells density: 2.10E05 cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): 6

GROWTH MEDIUM
- Standard medium used: Yes, as described in the guideline OECD 201.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Test medium was prepared using reverse osmosis purified deionised water.
- Intervals of water quality measurement: The pH of each control and test flask was determined at test start and test end. The temperature in the incubator was recorded daily.

OTHER TEST CONDITIONS
- Photoperiod: Continuous light
- Light intensity and quality: 7000 lux, 380 - 730 nm

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Determination of cell concentrations: At 0 and 28 h cell densities were determined using a Coulter Counter. At 50 and 72 h cell densities were determined using a haemocytometer and microscope as there were many small cells present that were not accurately counted by the Coulter Counter.

TEST CONCENTRATIONS
- Preliminary study:
- Test concentrations: control, 0.004 mg/L
- Results used to determine the conditions for the definitive study: No effect on algal growth was observed at the concentration of 0.004 mg/L.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.001 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: filtered test solution

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 0.001 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: filtered test solution

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

>= 0.001 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: filtered test solution

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 0.001 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: filtered test solution

Details on results:

- All validity criteria were fulfilled.
- Exponential growth in the control (for algal test): Yes, increase by factor of 54.
- Observation of abnormalities: Microscopical inspection showed no abnormalities of cells.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No
- Analytics: Measured concentrations were 0.00183 - 0.00185 mg/L at test start and 0.000552 - 0.00069 mg/L after 72 h. The geometric mean measured test concentration was determined to be 0.00107 mg/L.

Results with reference substance (positive control):

- Results with reference substance valid? Yes, the results were within the normal range.
- ErC50 (0 - 72 h): 0.58 mg/L, 95% CL: 0.47 - 0.71 mg/L
- EyC50 (0 - 72 h): 0.16 mg/L, 95% CL: 0.14 - 0.19 mg/L
- NOEC: 0.0625 mg/L
- Other: The positive control was conducted 28 Nov - 01 Dec 2006

Reported statistics and error estimates:

Statistical analysis of the growth rate data was carried out for the solvent control and 0.00107 mg/L test group using a Student’s t-test incorporating Bartlett’s test for homogeneity of variance. There were no statistically significant differences (p ≥ 0.05) between the solvent control and the 0.00107 mg/L test group and therefore the NOEC based on growth rate was 0.00107.

Validity criteria fulfilled:

yes

Conclusions:

The 72-hour EC50 and 72-hour NOEC on Desmodesmus subspicatus were > 0.00107 mg/L; showing that the test material is not harmful to algae.

Executive summary:

The toxicity to algae of the test substance was assessed according to the OECD 201 guideline and under GLP conditions.

 

Following a preliminary range-finding test, Desmodesmus subspicatus was exposed to an aqueous solution of the test material at a nominal concentration of 0.0040 mg/L (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 +/- 1 °C. Samples of the algal populations were removed daily and cell concentrations determined using a particle counter or a haemocytometer and light microscope.

Samples for analytical confirmation of exposure concentrations were taken at the start and after 72 hours of exposure. Since the measured concentration had declined at 72 hours, it was considered appropriate to base the results on the geometric mean measured tests concentrations.

 

The EC50 and the NOEC for growth rate inhibition and yield inhibition were beyond the range tested, i.e. exceeded the limit of solubility. The 72h-NOEC for growth rate and yield inhibition were superior or equal to 0.00107 mg/L. The test material is not harmful to algae both for short-term and long-term considerations.

Description of key information

NOEC (72h) >= 0.00107 mg/L (measured) for Desmodesmus subspicatus (OECD 201) 
No toxic effects occur in the range of the substance's water solubility. The test item was initially suspended in solubilizer. Therefore test concentrations above the solubility in aquatic test could be reached and even within these higher tested concentrations no toxic effects occurred.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

1 mg/L

Additional information

One study is available on the toxicity of 4,4´-(9H-fluoren-9-ylidene)bis(2-chloroaniline) (CAS No. 107934-68-9) to aquatic algae (Vryenhoef and Mullee, 2007). The study was performed according to OECD guideline 201 and GLP, with the unicellular freshwater green algae Desmodesmus subspicatus as test species. Since the substance has a low water solubility (< 0.052 mg/L) the test item was initially dissolved in dimethylformamide. The stock solution was stirred for 24 h using a magnetic stirrer and then filtrated (0.2 µm) to remove the undissolved test material. The test was conducted as limit test with a measured test substance concentration of 0.00107 mg/L (geometric mean). During the 72 hours test duration no effects on algae growth was observed.