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Ecotoxicological information

Long-term toxicity to aquatic invertebrates

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Description of key information

No adverse effects were observed at the limit of solubility

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater invertebrates:
0.35 mg/L

Additional information

A daphnia reproduction study with indigo is reported in the OECD SID document for Indigo Blue. The OECD guideline followed for this study was reported to be OECD test guideline 202 dated 1984, which is the test guideline for acute daphnia studies. No details on study conduct of test substance are available. Almost all concentrations tested (0.78 to 78 mg/L) were above the value for water solubility of 0.99 mg/L as stated in the OECD SIDS document. The test substance concentrations were tested in an extremely high concentration of the co-solvent DMSO:HCO-40 = 9:1 (1000 mg/L) without giving any information on solubility of Indigo in this solvent or effects seen in the vehicle control group. No analytical monitoring took place during the study. As DMSO has surface active properties, the use of high concentrations in the daphnia tests could effect the mobility and mortality of the daphnids, as surface active substances prevent the daphnids from diving and thus lead to the death of the daphnia. No information on effects of mobility of the daphnids were given in the OECD SIDS document. Hence, it is unknown whether the effects seen are due to the vehicle used or not, especially as they lie above the limit of water solubility for Indigo.

As a consequence, this study is deemed not reliable and cannot be used for assessment of the registered substance.

A second study (Bruns E, 2005) is available on reproductive effects on Daphnia magna conducted according to OECD test guideline 211 (Daphnia magna ( Reproduction Test; adopted: Sept. 21, 1998). This test was performed with a well-defined Indigo batch, which is in representative for the registered substance.

The dose levels used in this semi-static study were 0.0035 to 0.35 mg/L, which is lower than the water solubility of Indigo of 0.99 mg/L as reported in the OECD SIDS document (without providing the test method used). The Lead Registrant's own report for water solubility, conducted by BASF using a spectrophotometric test method, reports the water solubility of Indigo to be below the limit for quantification of 0.05 mg/L. Analytical monitoring (HPLC) took place throughout the study in the stock solution as well as the test vessels of the highest test concentration. Analytical monitoring of all other concentrations tested was not possible, as these concentrations were below the lower limit of quantification of the analytical test method used. No test substance-related effects were noted on mortality or reproduction rate at any concentration tested. The reproductive output of the Daphnia exposed to the test item was compared to that of the control in order to determine the Lowest Observed Effect Concentration (LOEC) and the No Observed Effect Concentration (NOEC) according to DUNNETT (1955, 1964). Additionally, the survival of the parent daphnia and the time to production of first brood was determined. 21 d NOEC (reproduction):       >= 0.35 mg/L

21 d LOEC (reproduction):        > 0.35 mg/L

21 d NOEC (mortality):             >= 0.35 mg/L

21 d LOEC (mortality):              > 0.35 mg/L

1st brood on day:                         5

The results are expressed in terms of nominal concentrations. Recovery rates ranged from 51.0 - 98.4% of the nominal values in the freshly prepared media and from 41.7 - 80.1% of nominal values in the media after 48 hours or 72 hours of exposure. It is hence concluded that Indigo has no effect on daphnia reproduction or survival after 21 days of exposure at the limit of water solubility.