Phenol, 2,4-dinitro-, sulfurized, leuco derivatives

Endpoint summary

Additional information

Alga

The influence of DIRESUL BLACK RDT LIQUID on the growth of the green algae Scenedesmus subspicatus was investigated. The study was performed according to the EEC Directive 92/69. The test method was modified to differentiate between a reduced growth of algae due to real toxic effects of the test substance on the algae cells or due to an indirect effect, a reduced algae growth by light absorption in coloured test solutions.

The test included two experimental parts: Experimental part A: the algae grew in test medium with dissolved dyestuff in Erlenmeyer flasks. Experimental part B: the algae grew in separate containers (test tube) which were surrounded by the black coloured solutions of the same concentration as tested in part A.

Because of difficulties concerning the solubility of DIRESUL BLACK RDT LIQUID in different test media, several tests were performed. Finally, in experimental part A the following nominal concentration range was tested: 0.01 mg/l to 200 mg/l. In experimental part B, the nominal concentration range of 0.01 mg/l to 50 mg/l was tested.

The biological results of both experimental parts A and B were almost identical. This was shown by several calculated parameters, the NOEC, LOEC and the EC 50 values:

In part A the NOEC was 1.0 mg/l, and in part B the NOEC was found to be between 1 and 5 mg/l. The LOEC was found to be between 1 and 5 mg/l in part A and it was about 5 mg/l in part B.

The percentage inhibition of algae biomass and the algae growth rate (IJ) after 72 hours of exposure were in the same order of magnitude. The EC 50 values calculated for both growth parameters, showed no significant differences.

Experimenta part A       EbC 50:       1.84 mg/l       Er C 50:       54.1 mg/l

Experimental Part B                           5.99 mg/l                          8.59 mg/l

Thus, the growth inhibition of Scenedesmus subspicatug was in the same magnitude when the algae grew in test medium 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 without dissolved test substance. In conclusion, this modified algae test clearly demonstrated that the observed inhibition effect of the substance was caused only by the indirect effect, the light absorption in the coloured test solutions. Thus, a toxic effect of the test substance on the algae cells can be excluded up to a concentration of 50 mg/l.

Fish, acute

The acute toxicity of the test item Leuco Sulfur Black 1 to zebra fish (Brachydanio rerio) was determined in a 96-hour semi-static test with a daily test medium renewal according to the Commission Regulation (EC) No 440/2008, Part C.1 and the OECD Guideline for Testing of Chemicals No. 203 (1992).

A limit test with a daily test medium renewal was performed in accordance with the test guidelines to demonstrate that the test item has no toxic effect on the test fish up to the loading rate of 100 mg/L.

Daphnia, acute

The acute toxicity of the test item Leuco Sulfur Black 1 to Daphnia magna was determined in a 48-hour semi-static test according to the EU Commission Directive 92/69/EEC, Part C.2, Commission Regulation (EC) No 440/2008, Part C.2 and the OECD Guideline for Testing of Chemicals, No. 202 (2004).

A limit test with a test medium renewal after 24 hours was performed in accordance with the test guidelines to demonstrate that the test item has no toxic effect on the test organisms up to the solubility limit of the test item in test water.

Due to the low water solubility of the test item, the test medium was prepared prior to the start of the test and prior to the test medium renewal as follows: a dispersion of the test item with the loading rate of 100 mg/L was ultrasonic treated for 15 minutes and continuously stirred at room temperature in the dark over 96 hours. Then, the dispersion was filtered. The undiluted filtrate of the dispersion was used as the test medium. Additionally, a control was tested in parallel. The preparation of the test medium was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substa Thences and Mixtures, 2000.

No remarkable observations were made concerning the appearance of the test medium. It was clear throughout the entire test duration.

No analytical work was performed due to the specific properties of the test item (insolubility in water).

At the beginning and end of the test medium renewal periods, the pH values of the test medium were between 7.6 and 8.0.

The dissolved oxygen concentrations in the test medium and control were at least 7.9 mg/L.

The water temperature was 20 °C during the test.

The biological results were based on the loading rate of the test item of 100 mg/L.

In the control and at the loading rate of 100 mg/L no immobilized daphnids were determined during the test period of 48 hours.

The biological test results (based on the loading rate) were as follows:

Loading rate (mg/L)

– 24-hour EC50: >100

– 24-hour EC0: ≥100

– 24-hour EC100: >100

– 48 -hour EC50: >100

– 48 -hour EC0: ≥100

– 48 -hour NOEC: ≥100

– 48 -hour EC100: >100

In conclusion, the test item Leuco Sulfur Black 1 had no acute toxic effects on Daphnia magna up to its solubility limit in the test water under the present test conditions.

Toxicity to bacteria

The inhibitory effect of the test item Leuco Sulfur Black 1 on the respiration rate of aerobic wastewater microorganisms of activated sludge was investigated in a 3-hour respiration inhibition test according to the EU Commission Directive 88/302/EEC, Part C.11, Commission Regulation (EC) No. 440/2008, Part C.11 and the OECD Guideline for Testing of Chemicals, No. 209.

Based on the EU testing guidelines, a limit test with a nominal test concentration of 1000 mg/L was performed.

In addition, two controls and three different concentrations of the reference item 3,5-dichlorophenol (5, 16, and 50 mg/L) were tested in parallel. The results of these treatments confirmed suitability of the activated sludge and the method used.

At the test item concentration of 1000 mg/L, the test item was not completely dissolved in the test medium. Thus, the test concentration was clearly above the water solubility limit of Leuco Sulfur Black 1 under the test conditions.

The test item had no inhibitory effect on the respiration rate of activated sludge after the incubation period of three hours at a loading rate of 1000 mg/L. The saturation concentration (water solubility limit of Leuco Sulfur Black 1 under the test conditions) was reached and no inhibitory effect was determined after three hours contact time.

Thus, the 3-hour NOEC (EC15) of the test item to activated sludge microorganisms was at least 1000 mg/L (i.e. the saturation concentration of the test item). This value might even be higher but concentrations above 1000 mg/L were not tested. The 3-hour EC20, EC50, and EC80 could not be calculated but were clearly higher than 1000 mg/L. Page