Trisodium 2,4-diamino-3,5-bis-[4-(2-sulfonatoethoxy)sulfonyl)phenylazo]benzenesulfonate

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:

February 21, 1997 to March 25, 1997

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

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

GLP compliance:

yes (incl. QA statement)

Remarks:

Swiss GLP

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: WP2/96
- Expiration date of the lot/batch: November 30, 2004

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature, in the dark
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: Solubility approximately 50 g/l (at 20 °C) and Stability for at least 48 h in water

Analytical monitoring:

yes

Details on sampling:

For the analytical measurements of the test substance concentrations duplicate samples were taken at the start of the test from the freshly prepared test media (without algae) of all test concentrations and from the control.
For the determination of the stability of the test substance under the test conditions, sufficient volumes of the freshly prepared test media of all test concentrations and the control were incubated under the same conditions as in the actual test (but without algae), and were sampled in duplicate at the end of the test (after the 72 h test period).
The concentrations of the test substance FAT 45'176/A were measured in the duplicate test media samples from all test concentrations and both sampling dates (0 and 72 h). From the control samples only one of the duplicate samples was analysed from each of both sampling dates (0 and 72 h).

Vehicle:

no

Details on test solutions:

The test medium of the highest test substance concentration was prepared by dissolving the test substance in test water (100 mg/l). Adequate volumes of this intensively mixed test medium were added to test water to prepare the test media of the following nominal concentrations: 1.0, 3.2, 10, 32 and 100 mg test substance/l. Additionally, a control was tested in parallel (test water without addition of the test substance). The test media were prepared just before the start of the test.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

The test organism used for the study was Scenedesmus subspicatus CHODAT, Strain No. 86.81 SAG, supplied by the "Sammlung von Algenkulturen, Pflanzenphysiologisches Institut der Universität Göttingen", D-37073 Göttingen. The algae had been grown in the laboratories of RCC under standardized conditions according to the test guidelines.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

0.24 mmol/l (= 24 mg/l) as CaCO3

Test temperature:

23-24 °C

pH:

7.9-8.5

Dissolved oxygen:

no data

Salinity:

no data

Nominal and measured concentrations:

nominal: 1.0, 3.2, 10, 32 and 100 mg test substance/l

Details on test conditions:

The test was started (0 hours) 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 about 72 hours prior to the test at the same conditions as in 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 with aluminium foil. The cylinders were covered with glass dishes, the dishes were covered with watch glass dishes to prevent evaporation.
The pH-values of the test media were measured in all test concentrations and the control at the start and the end of the test.
During the test duration the test media temperatures were measured daily in an Erlenmeyer flask filled with water and incubated under the same conditions as the test flasks.
Small volumes of the test media (1.0 ml) were taken out of all test flasks after 24, 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), three measurements per sample.
In addition, a sample was taken from the control and from the test concentration of nominal 32 mg test substance/l in experimental part A with reduced algal growth after the test period of 72 hours. The shape of these treated algal cells was microscopically examined and compared with the cells in the control.

Reference substance (positive control):

not specified

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

40.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

6.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

The test substance FAT 45'176/A was not stable in test water under the test conditions. The analytically determined test substance concentrations at the start of the test ranged from 97 % to 103 % of the nominal values, if the analytical measurements were based on parent compound and degradation products. During the test period of 72 hours a decrease of test substance concentration in the test media to 42 % - 94 % of the nominal values was determined even if based on the sum of parent compound and degradation products.
If the analytical measurements were based only on parent compound, at the start of the test 46 % to 55 % of the nominal values were found. At the end of the test no parent compound was found in test medium.
Since the analytically determined test substance concentrations at the start of the test, based on parent compound and degradation products were in good agreement with the nominal test concentrations (in the range from 97 % to 103 % of the nominal values) the reported biological results are related to the nominal test substance concentrations.

Results with reference substance (positive control):

no data

Reported statistics and error estimates:

The EbC 50 and EµC 50 (the concentrations of the test substance corresponding to 50 % inhibition of algal biomass respectively growth rate compared to the control), and the corresponding EC 10 and their 95 %-confidence limits were calculated for both experimental parts by Probit Analysis.
For the determination of the LOEC and NOEC, the calculated mean growth rates µ at the test concentrations were tested on significant differences to the control values by Dunnett-Tests.

Biological results:

Experimental part A

Experimental part A corresponds to the usual algal toxicity test. This means that the algal growth inhibition in this experimental part was caused by a possible toxic effect of the test substance and/or by the reduced light intensities due to the light absorption in the coloured test media.

In experimental part A, the test substance had a statistically significant inhibitory effect on the growth of Scenedesmus subspicatus after the exposure period of 72 h first at the concentration of nominal 3.2 mg test substance/1 (results of a Dunnett-test, one-sided, a = 0.05). Thus, this test concentration was determined as the 72-h LOEC (lowest concentration tested with toxic effects). The 72 -h NOEC (highest concentration tested without toxic effects after a test period of 72 h) was determined at the concentration of nominal 1.0 mg test substance/1, since at this test concentration the mean growth rates of the algae were statistically not significantly lower than in the control.

At the microscopical examination of the shape of the algal cells after 72 h incubation period no difference was observed between the algae growing in the test substance concentration of 32 mg test substance/1 in experimental part A and the algal cells in the control. Thus, the shape of the algal cells, growing in the test solution with this concentration of dissolved test substance was obviously not affected.

Experimental part B

In experimental part B the algal growth inhibition by the pure light effect (the reduced hght intensities in the coloured test media) was quantified. The EC-values in experimental part B were higher than the corresponding EC-values in experimental part A. Thus, only a part of the algal growth inhibition was obviously caused by the pure light effect. In experimental part B the algal growth was significantly reduced compared to the control after 72 h test period first at the test concentration of 32 mg test substance/l

Validity criteria fulfilled:

yes

Conclusions:

The 72 h EC50 of FAT 45176/A based on growth rate was above 100 mg/l while 72 h NOEC was 1.0 mg test substance/l.

Executive summary:

The influence of the test substance FAT 45' 176/A on the growth of the green algal species Scenedesmus subspicatus was investigated in a 72-h static test according to OECD 201 and EU C.3 guideline. However, 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 1.0, 3.2, 10, 32 and 100 mg test substance/l and a control. All test media down to the lowest test concentration were slightly to strongly coloured by the test substance.

The analytically determined test substance concentrations at the start of the test ranged from 97 % to 103 % of the nominal values, if the analytical measurements were based on parent compound and additionally on degradation products. During the test period of 72 h a decrease of test substance concentration in the test media to 42 % - 94 % of the nominal values was determined. If the analytical measurements were based only on parent compound at the start of the test 46 % to 55 % of the nominal values were found. At the end of the test no parent compound was found in test medium. Since the analytically determined test substance concentrations at the start of the test, based on parent compound and degradation products were in good agreement with the nominal test concentrations the reported biological results are related to the nominal test substance concentrations.

 

The growth inhibition effect of FAT 45'176/A on Scenedesmus subspicatus was caused in part due to the indirect effect, the light absorption in the coloured test solutions. However, a real toxic effect of the test substance on the growth of Scenedesmus subspicatus can not be excluded. Therefore, the results of this experimental part, where the algae grew in the test media with dissolved test substance as in a usual algal growth inhibition test should be taken into consideration for the determination of the toxic effect of the test substance on the growth of Scenedesmus subspicatus.

 

In this experimental part FAT 45' 176/A had a statistically significant inhibition effect on the growth of Scenedesmus subspicatus after the exposure period of 72 h first at the concentration of 3.2 mg test substance/l (= 72-h LOEC: lowest concentration tested with toxic effects). The 72-h NOEC (highest concentration tested without toxic effects) was 1.0 mg test substance/l, since at this test concentration the mean growth rates of the algae were statistically not significantly lower than in the control. The 72 h EC₅₀ based on growth rate was above100 mg/l.

Description of key information

The 72-h NOEC of Reactive Orange 136 was 1.0 mg test substance/l and the 72 h EC50 based on growth rate was above 100 mg/l.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

1 mg/L

Additional information

The influence of the test substance FAT 45' 176/A on the growth of the green algal speciesScenedesmus subspicatuswas investigated in a 72-h static test according to OECD 201 and EU C.3 guideline. However, 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 1.0, 3.2, 10, 32 and 100 mg test substance/l and a control. All test media down to the lowest test concentration were slightly to strongly coloured by the test substance.

The analytically determined test substance concentrations at the start of the test ranged from 97 % to 103 % of the nominal values, if the analytical measurements were based on parent compound and additionally on degradation products. During the test period of 72 h a decrease of test substance concentration in the test media to 42 % - 94 % of the nominal values was determined. If the analytical measurements were based only on parent compound at the start of the test 46 % to 55 % of the nominal values were found. At the end of the test no parent compound was found in test medium. Since the analytically determined test substance concentrations at the start of the test, based on parent compound and degradation products were in good agreement with the nominal test concentrations the reported biological results are related to the nominal test substance concentrations.

 

The growth inhibition effect of FAT 45'176/A onScenedesmus subspicatuswas caused in part due to the indirect effect, the light absorption in the coloured test solutions. However, a real toxic effect of the test substance on the growth ofScenedesmus subspicatuscan not be excluded. Therefore, the results of this experimental part, where the algae grew in the test media with dissolved test substance as in a usual algal growth inhibition test should be taken into consideration for the determination of the toxic effect of the test substance on the growth ofScenedesmus subspicatus.

 

In this experimental part FAT 45' 176/A had a statistically significant inhibition effect on the growth ofScenedesmus subspicatusafter the exposure period of 72 h first at the concentration of 3.2 mg test substance/l (= 72-h LOEC: lowest concentration tested with toxic effects). The 72-h NOEC (highest concentration tested without toxic effects) was 1.0 mg test substance/l, since at this test concentration the mean growth rates of the algae were statistically not significantly lower than in the control. The 72 h EC₅₀based on growth rate was above100 mg/l.