2,7-Naphthalenedisulfonic acid, 4-amino-3,6-bis[2-[4-(ethenylsulfonyl)phenyl]diazenyl]-5-hydroxy-, sodium salt (1:2)

Toxicity to aquatic algae and cyanobacteria

Description of key information

The 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.

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 test substance Reactive Black 5, the precursor of Reactive Black 5 bis-vinyl, 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.