Reaction mass of 6-Chloro-2-{4[ethyl(2-hydroxyethyl)amino]phenyl}-1-methylbenzo[cd]indolium hydroxide and 6-chloro-2-[4-[ethyl(2-hydroxyethyl)amino]phenyl]-1-methylbenz[cd]indolium acetate

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:

Test start date: 23 April, 1990; Test end date: 01 June 1990; Study completion date: 16 November, 1990.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Identity: FAT 31064/F
Batch: EN 158496.82 / HEW 133/6
Aggregate State: Powder
Solubility: Aqueous: >50 g/L at 20 °C
Org.solv.: acetone, alcohol, DMSO.DMF,
Chloroform: >2 g/1000 g at 20 °C
Appearance in the Test Medium: Violet solution (determined by RCC)
Stability: Aqueous medium: stable for at least 2 hours
Org. solvents: stable for at least 2 hours
Storage: At room temperature, in the dark
Expiration Date: November, 1994
Safety Precautions: Routine hygienic procedures were sufficient to assure personnel health and safety.

Analytical monitoring:

yes

Details on sampling:

Analysis of Samples

Control- 0.02 mg/l- and 0.313 mg/l Samples:
The total volume of the samples (5 ml) was mixed with 20 ml of bidistilled water and 3 ml of brine. This mixture was extracted twice with 25 ml of dichloromethane. The combined organic layers were dried on anhydrous sodium sulphate (pre-rinsed with dichloromethane). The latter was rinsed with 20 ml of dichloromethane. The combined dichloromethane phases were evaporated to dryness at 40 °C. The residue was dissolved in a mixture of acetonitrile-bidistilled wateracetic acid 25 ml-75 ml-1 ml. An aliquot (50 micro liter) of this solution was quantified by HPLC.

5.0 mg/l Samples:
The total volume of the samples (5 ml) was transferred into a 10 ml volumetric flask and filled to volume-with a mixture of acetonitrile-bidistilled water-acetic acid 50 ml-50 ml-1 ml. An aliquot (50 micro liter) of the diluted samples was quantified by HPLC.

Stability Test Samples 10 mg/l:
The 10 ml stability test samples were transferred into a 20 ml volumetric flask and the 100 ml stability test samples into a 200 ml volumetric flask. The flask were filled to volume with a mixture of acetonitrile-bidistilled water-acetic acid 50 ml-50 ml-1 ml.
The 100 ml stability test samples were further diluted ten times with thevmixture described before. An aliquot (50 micro liter) of the diluted samples was quantified by HPLC.

Vehicle:

no

Details on test solutions:

100 mg FAT 31064/F were made up to 100 ml with test medium and total dissolution effected by sonication with ultra sound. Thereafter, series of sequential dilutions with the test medium were prepared to obtain the final test concentrations of the test article which were based on pre-test results:
5, 1.25, 0.313, 0.078 and 0.020 mg/l.
An additional test concentration (10 mg/l) without algae was made and kept under the same test conditions as in the test. All tests were run in triplicate. In addition to the test treated samples, control samples were incubated.
As reference substance, Potassium dichromate was used.

Samples of 3.5 ml algae suspension were taken after about 24, 48, 72 and 96 hours of exposure. The number of algae after 24 and 48 hours was estimated by means of a microscope using a Neubauer chamber. After 72 and 96 hours, control samples and the three lower test concentrations (0.313, 0.078 and 0.020 mg/l) were determined spectro-photometrically on a Perkin-Elmer Lambda 15 by reading the adsorption at 700 nm and using a standard curve established with known (counted) cell density. For the test concentration of 1.25 and 5 mg/l, the number of algae after 72 and 96 hours was estimated by means of a microscope.

Test organisms (species):

Scenedesmus sp.

Details on test organisms:

The test organism used for the study was Scenedesmus subspicatus chodat supplied by the "Umweltbundesamt", Berlin (West), West Germany. The algae were grown in the laboratories of RCC under standardized conditions. The green algae scenedesmus subspicatus chodat is recognized by the OECD as species for the determination of the toxic effects of chemical compounds on the growth of planktonic freshwater algae.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Test temperature:

24 °C

pH:

The pH of the test medium was adjusted to 7.7 at the beginning of the tests. It was determined for all concentrations of the test article at the end of the incubation.

Nominal and measured concentrations:

5, 1.25, 0.313, 0.078 and 0.020 mg/L.

Details on test conditions:

Nutrient Solution: The algae were cultivated in a nutrient solution prepared according to OECD Guideline 201.

The experiments were started with a biomass of l000 cells per ml of nutrient solution. These cells were taken from a pre-culture which was set up 3 days prior to the test with the same conditions as the test.
Per test concentration, 30 ml of algae suspension were incubated in a shaking water bath (120 strokes/min.) at 24 °C with continuous illumination at 8000 Lux.
This illumination was achieved by four fluorescent tubes (Philips TL 33, 20 W,60 cm) installed above the shaking water bath in a distance from the algae flasks of about 20 cm. Incubation flasks used were 50 ml Erlenmeyer flasks stoppered with cotton wool plugs. The tests were run for 96 hours. Samples of algae were taken after about 24, 48, 72 and 96 hours of incubation.

Reference substance (positive control):

yes

Key result

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

0.507 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

When the EC50 was calculated by the Logit-Model, a value of EC50 = 0.507 mg/l was obtained with the following confidence limits:
90 % confidence limit: 0.488 - 0.526 mg/l
95 % confidence limit: 0.484 - 0.530 mg/l
99 % confidence limit: 0.477 - 0.537 mg/l

The dose response curve was found to be:
Logit = 0.82 + 1.207 x Log (dose).
The 95 % confidence limits for the slope were:
1.206 - 1.208.
The NOEC-value obtained was 0.078 mg/l.
The LOEC-value obtained was 0.313 mg/l.

Results with reference substance (positive control):

The results for the standard potassium dichromate are:
Based on the areas under the respective growth curves, the following EC 50 estimate for potassium dichromate was established (Logit): EC50 = 1.514 mg/l

90 % confidence limit: 1.492 - 1.537 mg/l
95 % confidence limit: 1.488 - 1.542 mg/l
99 X confidence limit: 1.48 - 1.551 mg/l
The dose response curve was found to be: Logit = -0.8203 + 1.977 x Log (dose).
The 95 % confidence limits for the slope were: 1.973 - 1.98

Reported statistics and error estimates:

The area under the growth curves (AUC) was calculated and the growth inhibition determined by comparison of the AUC-values of treated samples with those of untreated samples. An estimate of the EC50 including the confidence limits after 72 and 96 hours and the dose response lines were determined by Logit-analysis (1). EC 0, EC 50 and EC 100 were also calculated by linear regression of the values used for Logit-model. In addition, the NOEC and LOEC after 72 and 96 hours were statistically determined with the Dunnett-test.

Validity criteria fulfilled:

yes

Conclusions:

When FAT 31064/F was assessed for its acute toxicity to Scenedesmus subspicatus over 96 hours, the following results were obtained:
NOEC (72 hours):0.078 mg/L
LOEC (72 hours): 0.313 mg/L
EC50 (72 hours): 0.507 mg/L

Executive summary:

FAT 31064/F was tested for its toxic effects on the growth of green alga (Scenedesmus subspicatus). The study was performed according to the OECD Guideline 201 in a GLP-certified laboratory. In the study, following concentration were tested; 5, 1.25, 0.313, 0.078 and 0.020 mg/L. Anaytical samples were collected at 24, 48 and 72 hours to assess the stability of test substance in test medium. Potassium dichromate was used as a positive control. The test substance was found to be stable in the test solution. Based on the growth inhibition curve, the NOEC values for 72 and 96 hours were found to be 0.078 mg/l and 0.313 mg/L respectively. The EC50 values after 72 and 96 hours were 0.893 mg/L and 0.507 mg/L, respectively. The EC50 of potassium dichromate was found to be 1.514 mg/L

Description of key information

FAT 31064/F was tested for its toxic effects on the growth of green alga (Scenedesmus subspicatus). The study was performed according to the OECD Guideline 201 in a GLP certified laboratory. In the study, following concentration were tested; 5, 1.25, 0.313, 0.078 and 0.020 mg/L. Anaytical samples were collected at 24, 48 and 72 hours to assess the stability of test substance in test medium. Potassium dichromate was used as a positive control.

The test substance was found to be stable in the test solution. Based on the growth inhibition curve, the NOEC values for 72 and 96 hours were found to be 0.078 mg/l and 0.313 mg/L respectively. The EC50 values after 72 and 96 hours were 0.893 mg/L and 0.507 mg/L, respectively. The EC50 of potassium dichromate was found to be 1.514 mg/L

Key value for chemical safety assessment

EC50 for freshwater algae:

0.507 mg/L