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

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 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 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):

	Results [mg/L]
	PART A	PART B
EbC 50 (0-72h)	9.1	10.4
EbC 10 (0-72h)	2	2.3
EbC 90 (0-72h)	40.7	47.5

 

Growth rate µ:

	Results [mg/L]
	PART A	PART B
EµC 50 (0-72h)	25.5	28.1
EµC 10 (0-72h)	5.1	5.9
EµC 90 (0-72h)	127.8	134.5

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.

A study was performed to assess adverse effects of the test substance on the growth (=increase in cell density) and the growth rate(=rate of increase in cell density with time) of the planktonic freshwater algal species Desmodesmus subspicatus over several generations. The study was conducted in accordance with EEC Methods for Determination of Ecotoxicity Annex to Directive 92/69/EEC (O.J. No. L383A, 29.12.92) Part C, Method 3 'Algal inhibition test' which is in most parts equivalent to the OECD Guideline for Testing of Chemicals No. 201 'Alga, Growth Inhibition Test' and an instruction for the performance of a modified algal growth inhibition test published by Memmert&Knoel1 (RCC Umweltchemie, August 1992).

Exponentially growing algal cells were exposed for a period of 72 hours to a range of concentrations, nominally 0.29, 0.64, 1.4, 3.1, 6.8, 15 and 33 mg/L dissolved in water (part I). A second series (part II) of test vessels treated in the same way as the controls, but placed below glass vessels containing the same range of concentrations of the test substance as described above, but without algae, was set up to distinguish between algistatic (indirect effect caused by light absorption) and algicide (toxic) effects.

 

The cell densities were measured at 24 hour intervals. Inhibition of the algal population was measured as reduction in growth (index b) and growth rate (index r), relative to control cultures grown under identical conditions. Growth and growth rates were used to calculate a No Observed Effect Concentration and a Lowest Observed Effect Concentration according to DUNNETT (1955, 1964). The following values were determined:

 

	Results [mg/L]
	PART I	PART II
EbC 50 (0-72h)	1.8	1.9
ErC 50 (0-72h)	4.9	6.2
NOEC [b] [t 0,05]	0.29	0.29
LOEC [b] [t 0,05]	0.64	0.64
NOEC [r] [t 0,05]	0.64	0.29
LOEC [r] [t 0,05]	1.4	0.64

 

 

All results are expressed in terms of nominal concentrations. Measured concentrations ranged from 96.6 - 122.1% of nominal values at 0 hours, and from 88.4 - 112.0% of nominal values at 72 hours; respectively.

A comparison of the results for the parameter "percentage inhibition of the growth rate" gained in PART I and II of the study results in only one case in a difference exceeding 10%. For the nominal concentration 6.8 mg/L a difference of 11.1% was determined. The concentrations above (I5 mg/L) and below (3.1 mg/L) 6.8 mg/L resulted in respective values of 3.2% and 8.8%. No dose response relationship demonstrating an algaecide effect on the growth of algae has been observed. Therefore, the inhibition of growth observed in this study seemed to be caused by light absorption only.