Tetrasodium 4-amino-5-hydroxy-6-[[2-methoxy-5-[[2-(sulphonatooxy)ethyl]sulphonyl]phenyl]azo]-3-[[4-[[2-(sulphonatooxy)ethyl]sulphonyl]phenyl]azo]naphthalene-2,7-disulphonate

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

see read across justification in section 13

Reason / purpose for cross-reference:

read-across source

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

9.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

Experimental part A

Remarks on result:

other: 3.4 to 24.5

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

Experimental part A

Remarks on result:

other: 0.4 to 9.9

Duration:

72 h

Dose descriptor:

EC90

Effect conc.:

40.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

Experimental part A

Remarks on result:

other: 8.6 to 193.4

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

ca. 5.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

Experimental part A

Remarks on result:

other: 1 to 26

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

25.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

Experimental part A

Remarks on result:

other: 8.8 to 74.4

Duration:

72 h

Dose descriptor:

EC90

Effect conc.:

127.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

Experimental part A

Remarks on result:

other: 18.6 to 880.1

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

2.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

- Experimental part B

Remarks on result:

other: 0.5 to 11

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

10.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

- Experimental part B

Remarks on result:

other: 3.8 to 28

Duration:

72 h

Dose descriptor:

EC90

Effect conc.:

47.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

- Experimental part B

Remarks on result:

other: 9.7 to 233

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

5.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

- Experimental part B

Remarks on result:

other: 1.2 to 29.2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

28.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

- Experimental part B

Remarks on result:

other: 9.7 to 81

Duration:

72 h

Dose descriptor:

EC90

Effect conc.:

134.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

- Experimental part B

Remarks on result:

other: 19.7 to 917.4

Details on results:

All test media down to the lowest test concentration were little to strongly colored by the test substance.
In the control the cell density has increased from nominal 1x10E04 cells/ml at the start of the test (0 hours) to 68.06x10E04 cells/ml (mean value) after 72 hours by a factor of approximately 68. The biological results of both experimental parts A and B are identical. This is demonstrated by several calculated parameters, the NOEC/LOEC, the percentage growth inhibition rates, the algal densities after 72 hours and the EC values.
In both experimental parts, the LOEC (lowest concentration tested with a statistically significant inhibition effect) for both the algal biomass and the growth rate µ amounted to 100 mg test substance/L. The NOEC (highest concentration tested without significant inhibition effect) amounted to 3.2 mg/L.
In all test concentrations the percentage inhibition of algal biomass and the algal growth rate µ after 72 hours exposure period were in the same magnitude. Specially in the test concentration range with significant growth inhibition effect (10 to 100 mg/L) the inhibition rates were in good conformity.
In all test concentrations the mean algal cell densities experimental part A, determined after 72 hours were statistically not significant different from the cell densities in experimental part B (results of t-tests, two sided, p < 0 05).
Also at the EC values, calculated for both growth parameters, no significant differences were observable between the values in both experimental parts.

Results with reference substance (positive control):

NA

Reported statistics and error estimates:

Probit analysis
Dunnett test one sided at 0.05
t-test at 0.05

Validity criteria fulfilled:

yes

Conclusions:

The modified algal test has clearly demonstrated that the observed growth inhibition effect of the structural analogue Reactive Black 5 on Scenedesmus subspicatus was caused only due to the indirect effect, the light absorption in the coloured test solutions. Thus, a toxic effect of the test substance on the algal cells can be excluded up to a concentration of 100 mg/L.

Executive summary:

The influence of the structural analogue Reactive Black 5 on the growth of the green alga Scenedesmus subspicatus Chodat was investigated in a 72-hour static test according to the OECD Guideline No. 201, adopted June 7, 1984. However, the test method was modified to differentiate between a reduced growth of algae due to real toxic effects of the test substance on the algal cells or due to an indirect effect, a reduced algal growth by light absorption in coloured test solutions.

The test included two experimental parts:

·     Experimental part A: the algae grew in test-media with dissolved dyestuff in Erlenmeyer flasks, each placed in a black cylinder. The cylinders were covered with glass dishes, containing untreated test water in this experimental part.

·     Experimental part B: the glass dishes above the cylinders contained the coloured dyestuff solutions with the same five test concentrations as in Part A, however without algae. In the Erlenmeyer flasks below, the algae grew in test water without dyestuff (as in control), however under changed light conditions due to the filter effect of the coloured test solutions in the glass dishes.

The nominal test concentrations were 1.0, 3.2, 10.0, 32.0 and 100 mg test substance/L and a control. All test media down to the lowest test concentration were little to strongly coloured by the test substance. The biological results of both experimental parts A and B are identical. This could be demonstrated by several calculated parameters; the NOEC/LOEC, the percentage growth inhibition rates, the algal densities after 72 hours, and the EC-values. In both parts, the LOEC (lowest concentration tested with a statistically significant inhibition effect) for both the algal biomass and the growth rate µ amounted to 10 mg test substance/L, the NOEC (highest concentration tested without a significant inhibition effect) amounted to 3.2 mg/L.

In all test concentrations, the percentage inhibition of algal biomass and the algal growth rate µ after 72 hours exposure period were in the same magnitude. The mean algal cell densities in experimental part A, determined after 72 hours were statistically not significant different from the cell densities in experimental part B.

Also at the EC-values, calculated for both growth parameters, no significant differences are observable between the values in both experimental parts:

Biomass (determined as the area under the growth curve) in:

Experimental part A

- E_bC 50 (0-72 h)  9.1 mg/L

- E_bC 10 (0-72 h)  2 mg/L

- E_bC 90 (0-72 h)  40.7 mg/L

Experimental part B

- E_bC 50 (0-72 h)  10.4 mg/L

- E_bC 10 (0-72 h)   2.3 mg/L

- E_bC 90 (0-72 h)  47.5 mg/L

Growth rate µ in:

Experimental part A

- EµC 50 (0-72 h)  25.5 mg/L

- EµC 10 (0-72 h)  5.1 mg/L

- EµC 90 (0-72 h)  127.8 mg/L  

Experimental part B

- EµC 50 (0-72 h)  28.1 mg/L

- EµC 10 (0-72 h)  5.9 mg/L 

- EµC 90 (0-72 h)  134.5 mg/L

Thus, 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 (experimental part B) as in experimental part A, where the algae grew in test solutions with dissolved test substance. In conclusion, this modified algal test has clearly demonstrated that the observed growth inhibition effect of the test substance Reactive Black 5 on Scenedesmus subspicatus was caused only due to the indirect effect, the light absorption in the coloured test solutions. Thus, a toxic effect of the test substance on the algal cells can be excluded up to a concentration of 100 mg/L.

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

October to December 1992

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:

yes

Remarks:

modified to differetiate between toxic effects and indirect effects due to light absorption in coloured test subtrance

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

A stock solution was prepared just before the start of the test by dissolving the test substance in test water (1.0 g/l). Adequate amounts of the intensively mixed stock solution were added to test water to prepare the following nominal concentrations: 1.0, 3.2, 10.0, 32.0 and 100 mg/L. Additionally, a control (test water without any additions) was tested. The test concentrations were based on the results of a range-finding test.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Scenedesmus subspicatus
- Strain: 86.81 SAG
- Source (laboratory, culture collection): Sammlung von Algenkulturen, Pflanzenphysiologisches Institut der Universität Göttingen, Germany
- Method of cultivation: in synthetic test water according to OECD guideline 201

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

NA

Hardness:

no data

Test temperature:

Start: 23°C
Day 1: 23.3°C
Day 2: 23.6°C
Day 3: 23.8°C

pH:

start: 7.8 to 7.9
end: 8.0 to 10.3

Dissolved oxygen:

no data

Salinity:

NA

Nominal and measured concentrations:

1.0, 3.2, 10.0, 32.0 and 100 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type: closed (Erlenmeyer flask covered with glass dishes, covered with wath glass dishes
- Material, size, headspace, fill volume: glass, 100 mL, -, 50 mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
- Number of organisms: 10E04 cells/mL

The test was performed in Erlenmeyer flasks (100 ml), each with 50 ml algal suspension, continuously stirred by magnetic stirrers, 3 flasks per test concentration and 6 flasks in control Each Erlenmeyer flask was placed in a black cylinder, coated inside with 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
Experimental part A
The algae grew in test media with dissolved dyestuff in the Erlenmeyer flasks (5 test concentrations and a control). All glass dishes above the cylinders contained untreated test water. Thus, the inhibition of algal growth in this experimental part was caused due to a real toxic effect of the dyestuff and in addition to the reduced light intensities in the coloured test media in the Erlenmeyer flasks
Experimental part B
In this experimental part the glass dishes above the cylinders contained the colored dyestuff solutions with the same five test concentrations as in Part A, however without algae (3 replicates per test concentration). In the Erlenmeyer flasks below, the algae grew in test water without dyestuff (as in control), however under changed light conditions due to the filter effect of the colored test solutions in the glass dishes. Thus, the growth inhibition in part B was caused due to light absorption only. The depth of the test solutions in the glass dishes was 20 mm, i.e. half the depth of the test solutions in the Erlenmeyer flasks because the algae in the stirred test solutions stay in the statistical mean in this mean depth.
All flasks were incubated in a temperated water bath and continuously illuminated at a mean light intensity of 8174 Lux, range 7350 8100 Lux. The light intensity was measured just before the start of the test below the coating cylinders in those areas, where the Erlenmeyer flasks were placed in the test. This illumination was achieved by fluorescent tubes (universal white L 25, 36 W) installed in top of the algal flasks.
The test duration was 72 hours.

TEST MEDIUM / WATER PARAMETERS see attachment


- Determination of cell concentrations: Samples of 1 ml test solution were taken out of all flasks under sterile conditions after 24, 48 and 72 hours of exposure and not replaced. The algae cell densities in the samples were determined by counting with an electronical particle counter (AL CELLCOUNTER, Model 871), three measurements per flask and time. In addition, a sample was taken from the control and from a test concentration with reduced algal growth (nominal 32.0 mg/L) after a test period of 72 hours. The shape of the treated algal cells was microscopically examined and compared with the cells in the control.

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

9.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

Experimental part A

Remarks on result:

other: 3.4 to 24.5

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

Experimental part A

Remarks on result:

other: 0.4 to 9.9

Duration:

72 h

Dose descriptor:

EC90

Effect conc.:

40.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

Experimental part A

Remarks on result:

other: 8.6 to 193.4

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

ca. 5.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

Experimental part A

Remarks on result:

other: 1 to 26

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

25.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

Experimental part A

Remarks on result:

other: 8.8 to 74.4

Duration:

72 h

Dose descriptor:

EC90

Effect conc.:

127.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

Experimental part A

Remarks on result:

other: 18.6 to 880.1

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

2.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

- Experimental part B

Remarks on result:

other: 0.5 to 11

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

10.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

- Experimental part B

Remarks on result:

other: 3.8 to 28

Duration:

72 h

Dose descriptor:

EC90

Effect conc.:

47.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

- Experimental part B

Remarks on result:

other: 9.7 to 233

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

5.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

- Experimental part B

Remarks on result:

other: 1.2 to 29.2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

28.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

- Experimental part B

Remarks on result:

other: 9.7 to 81

Duration:

72 h

Dose descriptor:

EC90

Effect conc.:

134.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

- Experimental part B

Remarks on result:

other: 19.7 to 917.4

Details on results:

All test media down to the lowest test concentration were little to strongly colored by the test substance.
In the control the cell density has increased from nominal 1x10E04 cells/ml at the start of the test (0 hours) to 68.06x10E04 cells/ml (mean value) after 72 hours by a factor of approximately 68. The biological results of both experimental parts A and B are identical. This is demonstrated by several calculated parameters, the NOEC/LOEC, the percentage growth inhibition rates, the algal densities after 72 hours and the EC values.
In both experimental parts, the LOEC (lowest concentration tested with a statistically significant inhibition effect) for both the algal biomass and the growth rate µ amounted to 100 mg test substance/L. The NOEC (highest concentration tested without significant inhibition effect) amounted to 3.2 mg/L.
In all test concentrations the percentage inhibition of algal biomass and the algal growth rate µ after 72 hours exposure period were in the same magnitude. Specially in the test concentration range with significant growth inhibition effect (10 to 100 mg/L) the inhibition rates were in good conformity.
In all test concentrations the mean algal cell densities experimental part A, determined after 72 hours were statistically not significant different from the cell densities in experimental part B (results of t-tests, two sided, p < 0 05).
Also at the EC values, calculated for both growth parameters, no significant differences were observable between the values in both experimental parts.

Results with reference substance (positive control):

NA

Reported statistics and error estimates:

Probit analysis
Dunnett test one sided at 0.05
t-test at 0.05

Validity criteria fulfilled:

yes

Conclusions:

The modified algal test has clearly demonstrated that the observed growth inhibition effect of the structural analogue Reactive Black 5 on Scenedesmus subspicatus was caused only due to the indirect effect, the light absorption in the coloured test solutions. Thus, a toxic effect of the test substance on the algal cells can be excluded up to a concentration of 100 mg/L.

Executive summary:

The influence of the structural analogue Reactive Black 5 on the growth of the green alga Scenedesmus subspicatus Chodat was investigated in a 72-hour static test according to the OECD Guideline No. 201, adopted June 7, 1984. However, the test method was modified to differentiate between a reduced growth of algae due to real toxic effects of the test substance on the algal cells or due to an indirect effect, a reduced algal growth by light absorption in coloured test solutions.

The test included two experimental parts:

·     Experimental part A: the algae grew in test-media with dissolved dyestuff in Erlenmeyer flasks, each placed in a black cylinder. The cylinders were covered with glass dishes, containing untreated test water in this experimental part.

·     Experimental part B: the glass dishes above the cylinders contained the coloured dyestuff solutions with the same five test concentrations as in Part A, however without algae. In the Erlenmeyer flasks below, the algae grew in test water without dyestuff (as in control), however under changed light conditions due to the filter effect of the coloured test solutions in the glass dishes.

The nominal test concentrations were 1.0, 3.2, 10.0, 32.0 and 100 mg test substance/L and a control. All test media down to the lowest test concentration were little to strongly coloured by the test substance. The biological results of both experimental parts A and B are identical. This could be demonstrated by several calculated parameters; the NOEC/LOEC, the percentage growth inhibition rates, the algal densities after 72 hours, and the EC-values. In both parts, the LOEC (lowest concentration tested with a statistically significant inhibition effect) for both the algal biomass and the growth rate µ amounted to 10 mg test substance/L, the NOEC (highest concentration tested without a significant inhibition effect) amounted to 3.2 mg/L.

In all test concentrations, the percentage inhibition of algal biomass and the algal growth rate µ after 72 hours exposure period were in the same magnitude. The mean algal cell densities in experimental part A, determined after 72 hours were statistically not significant different from the cell densities in experimental part B.

Also at the EC-values, calculated for both growth parameters, no significant differences are observable between the values in both experimental parts:

Biomass (determined as the area under the growth curve) in:

Experimental part A

- E_bC 50 (0-72 h)  9.1 mg/L

- E_bC 10 (0-72 h)  2 mg/L

- E_bC 90 (0-72 h)  40.7 mg/L

Experimental part B

- E_bC 50 (0-72 h)  10.4 mg/L

- E_bC 10 (0-72 h)   2.3 mg/L

- E_bC 90 (0-72 h)  47.5 mg/L

Growth rate µ in:

Experimental part A

- EµC 50 (0-72 h)  25.5 mg/L

- EµC 10 (0-72 h)  5.1 mg/L

- EµC 90 (0-72 h)  127.8 mg/L  

Experimental part B

- EµC 50 (0-72 h)  28.1 mg/L

- EµC 10 (0-72 h)  5.9 mg/L 

- EµC 90 (0-72 h)  134.5 mg/L

Thus, 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 (experimental part B) as in experimental part A, where the algae grew in test solutions with dissolved test substance. In conclusion, this modified algal test has clearly demonstrated that the observed growth inhibition effect of the test substance Reactive Black 5 on Scenedesmus subspicatus was caused only due to the indirect effect, the light absorption in the coloured test solutions. Thus, a toxic effect of the test substance on the algal cells can be excluded up to a concentration of 100 mg/L.

Description of key information

The modified algal test has clearly demonstrated that the observed growth inhibition effect of the structural analogue Reactive Black 5 on Scenedesmus subspicatus was caused only due to the indirect effect, the light absorption in the coloured test solutions. Thus, a toxic effect of the test substance on the algal cells can be excluded up to a concentration of 100 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

100 mg/L

Additional information

The influence of the structural analogue Reactive Black 5 on the growth of the green alga Scenedesmus subspicatus Chodat was investigated in a 72-hour static test according to the OECD Guideline No. 201, adopted June 7, 1984. However, the test method was modified to differentiate between a reduced growth of algae due to real toxic effects of the test substance on the algal cells or due to an indirect effect, a reduced algal growth by light absorption in coloured test solutions.

The test included two experimental parts:

·     Experimental part A: the algae grew in test-media with dissolved dyestuff in Erlenmeyer flasks, each placed in a black cylinder. The cylinders were covered with glass dishes, containing untreated test water in this experimental part.

·     Experimental part B: the glass dishes above the cylinders contained the coloured dyestuff solutions with the same five test concentrations as in Part A, however without algae. In the Erlenmeyer flasks below, the algae grew in test water without dyestuff (as in control), however under changed light conditions due to the filter effect of the coloured test solutions in the glass dishes.

The nominal test concentrations were 1.0, 3.2, 10.0, 32.0 and 100 mg test substance/L and a control. All test media down to the lowest test concentration were little to strongly coloured by the test substance. The biological results of both experimental parts A and B are identical. This could be demonstrated by several calculated parameters; the NOEC/LOEC, the percentage growth inhibition rates, the algal densities after 72 hours, and the EC-values. In both parts, the LOEC (lowest concentration tested with a statistically significant inhibition effect) for both the algal biomass and the growth rate µ amounted to 10 mg test substance/L, the NOEC (highest concentration tested without a significant inhibition effect) amounted to 3.2 mg/L.

In all test concentrations, the percentage inhibition of algal biomass and the algal growth rate µ after 72 hours exposure period were in the same magnitude. The mean algal cell densities in experimental part A, determined after 72 hours were statistically not significantly different from the cell densities in experimental part B.

Also at the EC-values, calculated for both growth parameters, no significant differences are observable between the values in both experimental parts:

Biomass (determined as the area under the growth curve) in:

Experimental part A

- E_bC 50 (0-72 h)  9.1 mg/L

- E_bC 10 (0-72 h)  2 mg/L

- E_bC 90 (0-72 h)  40.7 mg/L

Experimental part B

- E_bC 50 (0-72 h)  10.4 mg/L

- E_bC 10 (0-72 h)   2.3 mg/L

- E_bC 90 (0-72 h)  47.5 mg/L

Growth rate µ in:

Experimental part A

- EµC 50 (0-72 h)  25.5 mg/L

- EµC 10 (0-72 h)  5.1 mg/L

- EµC 90 (0-72 h)  127.8 mg/L  

Experimental part B

- EµC 50 (0-72 h)  28.1 mg/L

- EµC 10 (0-72 h)  5.9 mg/L 

- EµC 90 (0-72 h)  134.5 mg/L

Thus, 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 (experimental part B) as in experimental part A, where the algae grew in test solutions with dissolved test substance. In conclusion, this modified algal test has clearly demonstrated that the observed growth inhibition effect of the test substance Reactive Black 5 on Scenedesmus subspicatus was caused only due to the indirect effect, the light absorption in the coloured test solutions. Thus, a toxic effect of the test substance on the algal cells can be excluded up to a concentration of 100 mg/L.

The same is considered to be true with the target substance Reactive Blue 250.