3-(2,4-bis(4-((5-(4,6-bis(2-aminopropylamino)-1,3,5-triazin-2-ylamino)-4-hydroxy-2,7-disulfonaphthalen-3-yl)azo)phenylamino)-1,3,5-triazin-6-ylamino)propyldiethylammonium lactate

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:

From July 8th to 11th,1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

December 29th,1992

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

June 7th, 1984

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Vehicle:

no

Details on test solutions:

The nominal concentrations tested were 3.9, 9, 19, 41, 91 and 200mg/l of the test substance. Additionally, a control group (test water without addition of the test substance) was tested in parallel. The analytical measurements of the test substance were performed in the test concentrations of 91 and 200 mg/l. The mean measured test substance concentrations (calculated as the average over the two measurements per test concentrations) amounted to 72 % and 81 %, respectively. Therefore, the biological results are based on the mean measured test substance concentrations.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

3 replicates per test concentration.
6 replicates for the test control.
At the start of the experiment (0 hours) a biomass of 10.000 algal cells was inoculated per ml of test medium. A volume of 15 ml algal suspension per replicate was continuously stirred bymagnetic stirrers in 50 ml Erlenmeyer flasks. The flasks were incubated in a temperature controller water bath and continuously illuminated at the measured light intensity of about 7700 Lux.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Hardness:

24 mg CaCO3/l

Test temperature:

The test media temperatures ranged from 22.0 to 22.4 °C.

pH:

The measured pH-values in the test media ranged from pH 7.5 to pH 8.2 at the start of the test and from pH 7.9 to 8.7 at the end of the test. .

Nominal and measured concentrations:

Nominal concentrations of the test substance = 3.9, 9, 19, 41, 91, 200 mg/l.
The analytical measurements of the test substance were performed in the test concentrations of 91 and 200 mg/l. The mean measured test substance concentrations (calculated as the average over the two measurements per test concentrations) amounted to 72 % (65 mg/l) and 81 % (161 mg/l), respectively. Therefore, the biological results are based on the mean measured test substance concentrations.

Details on test conditions:

In the control the cell density has increased from nominal 1 × 10^4 cells/ml at the start of the test (0 hours) to 75.52 × 10^4 cells/ml (mean value) after 72 hours by a factor of approximately 73. Thus, the algal growth in the control was sufftciently high under the test conditions.

In Milli-Q-water salts were added: NaHCO3 (50.0 mg/l), CaCl2 × 2H2O (18.0 mg/l), NH4Cl (15.0 mg/l), MgSO4 × 7H2O (15.0 mg/l), MgCl2 × 6H2O (12.0 mg/l), KH2PO4 (1.6 mg/l).

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 161 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 161 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

65 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

65 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

161 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

The analytical measurements of the test substance were performed for the test concentrations of 91 and 200 mg/l. At the end of the test, 57 % and 68 %, respectively, of the nominal values were found. The losses could be due to a degradation of the test substance as a consequence of the intense irradiation of the samples.
The test substance had a statistically significant inhibitory effect on the growth of Scenedesmus subspicatus after the exposure period of 72 hours at the concentration of the test substance of 161 mg/l (nominal 200 mg/l). The growth rate was slightly, but statistically significant, reduced also at the nominal test concentration 41 mg/l. However, at the next higher test concentration of 65 mg/l (nominal 91 mg/l) the growth rate was not significantly reduced in comparison to the control after 72 hours. Thus, the test substance concentration of 161 mg/L (nominal 200 mg/l) was determined as the 72-hour LOEC. The test media of all test concentrations were coloured by the test substance. The slight inhibition effect of the test substance at the highest test concentration of 161 mg/l might even be caused by pure light reduction in the coloured test medium.
The 72-hour NOEC was determined at the test substance concentration of 65 mg/l (nominal 91 mg/l), since up to and including this test concentration both the mean biomass and the mean growth rates of the algae were statistically not significant lower than in the control.
The test concentrations corresponding to 10, 50 and 90 % inhibition of the algal biomass (EC10, 50, 90) and of the growth rate could not be calculated due to the low growth inhibition effect up to the hightest concentration. The 72-hour EC 50-biomass and the 72-hour EC 50-growth rate were clearly higher than 161 mg/I.
At the microscopic examination of the shape of the algal cells after 72 hours test period no difference was observed between the algae growing in the test substance concentration of nominal 91 mg/L and the algal cells in the control. Thus, the shape of the algal cells growing at least up to this test concentration was obviously not affected.

Conclusions:

EC10, EC50, EC90 (72h) for algal biomass and growth rate could not be calculated due to the low growth inhibition effect up to the highest test concentration.

Executive summary:

The influence of the test item on the growth of the green algal species Scenedesmus subspicatus was investigated in a 72-hour static test according to EU Method C.3 (Algal Inhibition test), and the OECD Guideline No.201, 1984. 

The test substance had a statistically significant inhibitory effect on the growth of Scenedesmus subspicatus after the exposure period of 72 hours at the concentration of the test substance of 161 mg/l (nominal 200 mg/l). The growth rate was slightly, but statistically significant, reduced also at the nominal test concentration 41 mg/l. However, at the next higher test concentration of 65 mg/l (nominal 91 mg/l) the growth rate was not significantly reduced in comparison to the control after 72 hours. Thus, the test substance concentration of 161 mg/L (nominal 200 mg/l) was determined as the 72-hour LOEC. 
The 72-hour NOEC was determined at the test substance concentration of 65 mg/l (nominal 91 mg/l).
The test concentrations corresponding to 10, 50 and 90 % inhibition of the algal biomass (EC10, 50, 90) and of the growth rate could not be calculated due to the low growth inhibition effect up to the hightest concentration. The 72-hour EC 50-biomass and the 72-hour EC 50-growth rate were clearly higher than 161 mg/I.

Description of key information

EC50 growth rate > 161 mg/L

Key value for chemical safety assessment

EC50 for freshwater algae:

161 mg/L

Additional information

The influence of the test item on the growth of the green algal species Scenedesmus subspicatus was investigated in a 72-hour static test according to EU Method C.3 (Algal Inhibition test), and the OECD Guideline No.201, 1984. 

The test substance had a statistically significant inhibitory effect on the growth of Scenedesmus subspicatus after the exposure period of 72 hours at the concentration of the test substance of 161 mg/l (nominal 200 mg/l). The growth rate was slightly, but statistically significant, reduced also at the nominal test concentration 41 mg/l. However, at the next higher test concentration of 65 mg/l (nominal 91 mg/l) the growth rate was not significantly reduced in comparison to the control after 72 hours. Thus, the test substance concentration of 161 mg/L (nominal 200 mg/l) was determined as the 72-hour LOEC. 
The 72-hour NOEC was determined at the test substance concentration of 65 mg/l (nominal 91 mg/l).
The test concentrations corresponding to 10, 50 and 90 % inhibition of the algal biomass (EC10, 50, 90) and of the growth rate could not be calculated due to the low growth inhibition effect up to the hightest concentration. The 72-hour EC 50-biomass and the 72-hour EC 50-growth rate were clearly higher than 161 mg/I.