Sodium 3-[(1,5-dihydroxy-2-naphthyl)azo]-4-hydroxybenzenesulphonate

Toxicity to aquatic algae and cyanobacteria

Description of key information

The substance forms complexes with metal ions; therefore, the algae toxicity cannot be determined

Additional information

The substance forms complexes with metal ions; therefore, the algae toxicity cannot be determined

A study was performed to assess the adverse effects of Mordant Black 9 on the growth rate (= rate of increase in cell density with time) and the yield (= biomass at time t minus initial biomass) of the planktonic freshwater algal species Desmodesmus subspicatus(former name: Scenedesmus subspicatus) over several generations.

 

The study was conducted in accordance with Commission Regulation (EC) No 761/2009 amending Regulation No 440/2008, Method C.3 ‘Freshwater Alga and Cyanobacteria, Growth inhibition test’ (2009) which is equivalent to OECD Guideline for Testing of Chemicals No. 201 (2006).

 

Exponentially growing algal cells were exposed for a period of 72 hours to a range of concentrations, nominally 0.01, 0.032, 0.1, 0.32, 1.0, 3.2, 10 and 32 mg/L of Mordant Black 9 dissolved in dilution water. Auxiliaries used to prepare the test media were an ultrasonic bath and a magnetic stirrer.

 

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

 

Results [mg/L]:   ErC 50* (0-72 h):            0.46 ErC 10* (0-72 h):            0.22 NOEC [r]* (tα 0.05):          0.019 LOEC [r]* (tα 0.05):           0.062

According to the data of the sponsor Mordant Black 9 is a complex forming dye and it is possible that Mordant Black 9 forms complexes with the essential metal ions contained in the algae growth medium, which are essential for the growth of the algae. So it is conceivable that the result of this alga, growth inhibition test is not due to the chemical toxic effect of Mordant Black 9 but is rather caused by the lack of essential elements. Therefore the results should be handled with care.

* Reduction of growth rate (E_rCx, NOEC [r]) is the preferred endpoint according to OECD 201 and for regulatory purposes in the EU. Results relating to yield (E_yCx, NOEC [y]) were calculated to fulfil regulatory requirements in some countries (but not in the EU) and are given in the results section of this report.

The results are expressed in terms of geometric mean measured concentrations. Effective concentrations ranged from 48.8 % to 80.0 % of nominal values at 0 hours, from 7.6 % to 10.8 % of nominal values at 24 hours, and from to 0.1 % to 3.2 % of nominal values at 48 hours and correspond to 0.7 % of nominal values at 72 hours. After 72 hours only the highest test item concentration was analysed, all other concentrations were below the quantification limit (0.0292 mg/L).

 

To reduce shading effects of the coloured test substance, the test was carried out with strong illumination (120-150 µE x m-2 x s-1) and with a reduced test volume of 25 ml in 300 mL Erlenmeyer flasks.

Mordant Black 9 is a complex forming dye, according to the data of the sponsor. The dye is stabilised on the fabric by adding chromium after applying the dye to the fabric to form a chromium complex. Due to this property as strong sequestrant, it is also conceivable that Mordant Black 9 forms complexes with the various metal ions contained in the algae growth medium such as Fe3+, Mn2+, Zn2+, Co2+, Cu2+. This is particularly critical with the trace elements, which are essential for the growth of the algae.

This effect is proven by the results of the substance concentration analysis. The results are expressed in terms of geometric mean measured concentrations. Effective concentrations ranged from 48.8 % to 80.0 % of nominal values at 0 hours (after 24 hours of stirring), from 7.6 % to 10.8 % of nominal values at 24 hours, and from to 0.1 % to 3.2 % of nominal values at 48 hours and correspond to 0.7 % of nominal values at 72 hours. After 72 hours only the highest test item concentration was analysed, all other concentrations were below the quantification limit (0.0292 mg/L).

That this effect is due to a complex formation has been shown by the evaluation of the IR spectrum of the original test item Mordant Black 9, the test item dissolved in millipore water and the test item dissolved in algae growth medium. The IR spectrum of the original test item Mordant Black 9 and of the test item dissolved in millipore water are identical, whereas the IR spectrum of Mordant Black 9 dissolved in algae growth medium differs distinctly from the former two.

Any growth inhibition of algae observed is therefore most likely due to the lack of essential elements and not due to a direct toxic effect of Mordant Black 9. This situation would not occur in the environment, where those trace elements are abundantly available.

Furthermore, if this complex forming feature of Mordant Black 9 takes place in the OECD medium, it is technically not possible to test just the chemical toxicity of the test substance on algae growth according to OECD 201.