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

Endpoint summary

Additional information

One study on alga (Stahl) was performed on similar substance 1 following EU C3 with an EC 50 at 72 based on Grow rate of 13.7 mg/l. It was not possible to recover the original report of the study and the few available details are reported Another study on alga (Reach&colours KFT, 2011) was performed on similar substance 2 following a modified version of OECD 201. The EC0 is equal to 1 mg/l and EC50 is between 10 and 100 mg/l (nominal). No effect on the algae toxicity is due to the counter ion and it is mainly due to the shadowing effect of the substance in the tested medium. Several studies on algae conducted on dark dyes, including those with a modified test system for dye-stuffs, showed that the growth inhibition is not due to a toxic effect of the dye, but due to the light absorption of the stained water. Modified test system is usually conducted putting the dye above the algae testing solution, in a different vessel and not into contact with the alga. The same toxicity expressed as grow rate and yield inhibition has been observed like when in the same condition the algae is into contact with the dye. It has been deduced that the observed toxicity was related to the shading effect of the dye. This method has some limitation because it focuses on the shadow effect but gives no information on the real potential toxicity for algae of the tested substance.

 

One test on Lemna (Reach&colours, 2012) has been conducted following OECD 221 on similar substance 1 using Lemna minor as aquatic plant. Lemna is an aquatic plant that develops his leaves on the surface of the water, while nourishing substances are taken from the water solution. With this test the observed effect is only related to the potential toxicity of the substance and not to the potential shading effect of a classical Alga study. A deviation to the protocol has been applied to the test recommended for dyes (Michael Cleuvers, Hans-Toni Ratte, Phytotoxicity of coloured substances: is Lemna Duckweed an alternative to the algal growth inhibition test? Chemosphere 49 (2002) 9–15): "Beakers will be incubated on a black non-reflecting surface. Additionally, the walls of the incubation chambers will also be covered with black fabric in order to avoid reflection". The EU authorities’ agreement refers to the Manual of Decision (EU Manual of Decisions dated July 2006, athttp://ecb.jrc.ec.europa.eu/new-chemicalssection 13.5.3 Alternatives to the algae growth inhibition test with coloured substances.)“where it can be demonstrated in the case of highly coloured substances that algal growth is inhibited solely as a result of a reduction in light intensity, then the 72h EC50 for algae should not be used as a basis for classification.” In order to make a distinction between shading and toxic inhibition effects, in 1994 some modifications to the algae growth inhibition test, known as the ETAD (Ecological and Toxicological Association of Dyes and Organic Pigments Manufacturers) method (also known as the double flask method), were suggested. Advanced insights revealed that the ETAD method is too simplistic to allow evaluation of both toxic and light absorption effects of the coloured substances and should not be used any more. For highly light absorbing substances, the modified standard algae growth inhibition test is not recommended. With these particular substances, a modified standard Lemna-test (draft OECD guideline 221) is recommended. The following modification to the standard Lemnat est has to be applied: the test has to be performed on a black, non-reflecting surface. While for the same substance the “classical alga” test has provided a result of EC50 of 45.3 mg/l, with Lemna no toxicity up to the maximum tested level of 2000 mg/l on the active substance has been observed. Therefore the toxicity to aquatic plants of the substance can be assessed at 2000 mg/l and the already performed studies on algae can be disregarded.

 

A limit test on Daphnia Magna has been performed on similar substance 1, showing no mortality at the tested concentration of 180 mg/L. Since the sample concentration is 83% of the nominal concentration, the EC50 has been set at 150 mg/L

Based on the read across justification the same values can be used for Acid Green 68. The similarity between the substances, the molecular formula and the toxicological and eco-toxicological considerations are reported in the attached document in section 13.