trisodium 4-hydroxy-6-(sulfonatomethylamino)-5-(2-(2-sulfatoethylsulfonyl)phenylazo)naphthalene-2-sulfonate

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:

1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

duplicates taken for UV/VIS spectroscopy

Vehicle:

no

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

Scenedesmus subspicatus Strain No.: 86.81 SAG supplied by "Sammlung von Algenkulturen, Pflanzenphysiologisches institut" Gottingen University. The algae had been grown in the RCC laboratories under standardized conditions according to the test guidelines.

Test type:

static

Water media type:

freshwater

Remarks:

synthetic test water prepared according to the mentioned guidelines

Limit test:

no

Total exposure duration:

72 h

Hardness:

0.24 mmol/L (24 mg/L) as CaCO3

Test temperature:

24 °C

pH:

At the begining of experiment: 8.0-8.1, at the end: 8.3-8.7.

Nominal and measured concentrations:

The nominal test concentrations were: 4.6; 10; 22; 46 and 100 mg/L and a control.

Details on test conditions:

The test was started by inoculation of a biomass of 10.000 algal cells per ml test medium. These cells were taken from an exponentially growing pre-culture, which was set up three days prior the test.
The test was performed in Erlenmeyer flasks (50 ml), each with 15 ml algal suspension, continuously stirred by magnetic stirrers, 3 flasks per test concentration and 6 flasks in the control. Each Erlenmeyer flask was placed in a black cylinder, coated inside by aluminum foil. The cylinders were covered with glass dishes, the dishes were covered with watch glass dishes to prevent evaporation.

The test included two experimental parts:
A:
The algae grew in test media with dissolved test substance in the Erlenmeyer flasks (five test concentrations and a control). All glass dishes above cylinders contained purified water. Thus, the inhibition of algal growth in this part was caused due to real toxic effect of the test substance and in addition to reduced light intensities in the colored test media in the Erlenmeyer flasks.
B:
In this part glass dishes above the cylinders contained the colored test media with the same concentrations as in part A, however without algae (3 replicates per test concentration). In the Erlenmeyer flasks below, the algae grew in the water without test substance (as in control), however under changed light conditions due to the filter effect of the colored test media in the glass dishes. Thus the growth inhibition in part B was caused due to light absorption only. The depth of the test media in the glass dishes was 4 mm – half of the depth of the media in Erlenmeyer flasks. All flasks were incubated in a temperature controlled water bath and continuously illuminated at a mean light intensity of about 7500 Lux (fluorescent tubes).

Counting and Examination of the algal cells:
Small volume of the test media (1.0-2.0 mL) were taken out of all test flasks after 2, 48 and 72 hours of exposure and were not replaced. The algae cell densities in the samples were determined by counting with an electronic particle counter (Coulter Counter, Model ZM), two measurements per sample. In addition, a sample was taken from the control and from the test concentration of nominal 46 mg test article/L in experiment A with reduced algal growth after the period of 72 hours. The shape of these algal cells was microscopically examined and compared with the cells in the control.

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC50

Remarks:

Experiment A

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Remarks:

Experiment A

Effect conc.:

16 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence intervals

Remarks:

(4.3-29.7)

Key result

Duration:

72 h

Dose descriptor:

EC50

Remarks:

Experiment A

Effect conc.:

106 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% confidence intervals

Remarks:

(49.3-1348)

Key result

Duration:

72 h

Dose descriptor:

EC10

Remarks:

Experiment A

Effect conc.:

3.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% confidence intervals

Remarks:

(0.1-9.1)

Key result

Duration:

72 h

Dose descriptor:

EC50

Remarks:

Experiment B

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Remarks:

Experiment B

Effect conc.:

14.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence intervals

Remarks:

(11.4-18.0)

Key result

Duration:

72 h

Dose descriptor:

EC50

Remarks:

Experiment B

Effect conc.:

51.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% confidence intervals

Remarks:

(42.0-65.9)

Key result

Duration:

72 h

Dose descriptor:

EC10

Remarks:

Experiment B

Effect conc.:

3.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% confidence intervals

Remarks:

(2.3-5.1)

Details on results:

Experiment A:
In experiment A the test substance had statistically significant inhibitory effect on the growth rate of Scenedesmus subspicatus after the exposure period of 72 hours first at the concentration of 10 mg/L (Dunnett test). At the concentration of 4.6 mg thest substance/L the mean growth rates of the algae were statistically not significantly lower than in the control.
At a microscopical examination of the shape of the algal cells after 72 hours incubation period no difference was observed between the algae growing in the test substance concentration of 46 mg/L in experiment A and the algal cells in the control.

Experiment B:
In experiment B the algal growth was statistically reduced compared to the control after 72 hours at the concentration of 10 mg/L.

Validity criteria fulfilled:

yes

Conclusions:

The growth rate and biomass growth were determined as follows, in experiment A, growth rate: EC10: 16.0 mg/L; EC50: >100 mg/L; biomass: EC10: 3.6 mg/L; EC50: 106 mg/L. In experiment B, growth rate: EC10: 14.7 mg/L; EC50: >100 mg/L; biomass: EC10: 3.7 mg/L; EC50: 51.4 mg/L. The substance is not acutely or chronically toxic to freshwater green algae. Any inhibitoty effect observed during this test was indirect due to light absorption (algistatic). The substance has no algicidal effect.

Executive summary:

A study was conducted to determine the toxicity of the test substance on the growth of the green algal species Scenedesmus subspicatus in a 72 h static test according to modified OECD 201 Guideline and EU Method C.3, in compliance with GLP. The test method was modified to quantify the algicidal effect of the test substance, but also the growth inhibition effect caused by reduced light intensities in the coloured test solutions. The nominal test concentrations were equivalent to 0, 4.6, 10, 22, 46 and 100 mg/L. All test media down to the lowest test concentration were slightly too strongly coloured by the test substance. The analytically determined mean concentrations in the analysed test media varied from 91 to 100% of nominal values. The total mean measured concentrations (calculated as the average over all measurements per test concentration) ranged from 92 to 98% of nominal values. The test substance was sufficiently stable in a media under the study conditions during the test period of 72 h. Therefore, all biological results were related to nominal concentrations. The same growth inhibition of Scenedesmus subspicatus was observed when the algae grew in test water without test substance, but under reduced light intensities by the filter effect of coloured test media as the second parallel experimental part, where the algae grew in the test media with dissolved test substance. Therefore, the observed growth inhibition was only due to an indirect effect, i.e. the light filter effect in the coloured test solutions. Thus, a toxic effect of the test substance on the algal cells can be excluded up to the highest test concentration of 100 mg/L. The growth rate and biomass growth were determined as follows, in experiment A, growth rate: EC10: 16.0 mg/L; EC50: >100 mg/L; biomass: EC10: 3.6 mg/L; EC50: 106 mg/L. In experiment B, growth rate: EC10: 14.7 mg/L; EC50: >100 mg/L; biomass: EC10: 3.7 mg/L; EC50: 51.4 mg/L (Memmert, 1998).

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L