reaction product of: C.I. Leuco Sulfur Black 1 with (3-chloro-2-hydroxypropyl)trimethylammonium chloride

Endpoint summary

Description of key information

LC50 (96 hours, Oncorhynchus mykiss) = 4.6 mg/l (nominal)

EC50 (48 hours, Daphnia magna) = 49 mg/l (mean measured)

ErC50 (72 hours, Scenedesmus subspicatus) = 14.6 mg/l (mean measured)

EC50 (30 minutes, aerobic waste water bacteria) > 100 mg/l (nominal)

Additional information

Toxicity to Fish

The toxicity of the test item to fish was evaluated in an experimental study on Rainbow trout (Oncorhynchus mykiss) according to the OECD Guideline 203 (1992) and the EU Method C.1 (1992). Young Rainbow trout were exposed to various concentrations of the test item in aqueous test media for 96 hours in a static test under defined conditions, and were monitored daily for mortality and symptoms of intoxication. The nominal concentrations of test item were 0.92, 2.0, 4.2, 9.2 and 20.0 mg/l. Aquaria were monitored for oxygen concentration, pH level, temperature and behaviour of the test item in test media.

The mean measured test substance concentrations were determined to be 90 % to 97 % of the nominal values (calculated as the average over all measurements per test concentration). Since these mean measured test substance concentrations were well above 80 % of the nominal values, the reported biological results are related to the nominal test substance concentrations.

Strong colouration was observed at all time points and at all concentrations, caused by the test item, therefore toxicity and symptoms were difficult to monitor. No mortality or toxicity was observed among the control group or test groups at concentrations of 0.92 and 2.0 mg/l test item, therefore the NOEC for 96 hours was determined to be 2.0 mg/l (LOEC = 4.2 mg/l). The LC50 was determined to be 4.6 at 96 hours (C.I. 95%: 3.0 - 7.0).

Toxicity to Aquatic Invertebrates

The toxicity of the test item to invertebrates was evaluated in an experimental study on Daphnia magna according to the OECD Guideline 202 (1984) and the EU Method C.2 (1992). Four groups of 5Daphniaper flask were exposed to various concentrations of the test item in aqueous test media for 48 hours in a static test under defined conditions, and were monitored daily for imobilisation. The nominal concentrations of test item were 9.2, 20, 42, 92 and 200 mg/l. Flasks were monitored for oxygen concentration, pH level, and behaviour of the test item in test media at the beginning and end of the study period.

In the test media the measured test substance concentrations at the start of the test ranged from 81 % to 98 % of the nominal values. During the test period of 48 hours a slight decrease of test substance concentration in the test media was determined. The mean measured test concentrations (calculated as the average over all measurements per test concentration) varied in the range of 76 % to 96 % of the nominal values. Therefore, the reponed biological results are related to the mean measured test substance concentrations.

Strong colouration of the test media and the Daphnia was observed, caused by the test item. Dissolved oxygen concentration ranged from 8.4 to 10.2 mg/l during the study period. Based on the observations, the EC50 (48 hours) was determined to be 49 mg/l test item at a 95 % confidence interval from 33 to 74 mg/l.

Toxicity to Aquatic Algae and Cyanobacteria

The toxicity of the test item to algae was evaluated in an experimental study onScenedesmus subspicatusaccording to the OECD Guideline 201 (1984) and the EU Method C.3 (1992). The test method was modified to quantify the algicidal effect of the test item, but also the growth inhibition effect due to the reduced light intensities in the coloured test media. 10,000Scenedesmus subspicatusper ml of test media were cultured and inoculated, then allowed to grow in 50 ml Erlenmeyer flasks containing 15 ml algal suspension, in triplicate, at test item concentrations of 1.9, 6.1, 19.5, 62.5 and 200 mg/l for 72 hours in a temperature-controlled, continuously lit water bath. A control containing no test item (six replicates) was also tested in parallel. Cell densities were recorded at 24, 48 and 72 hours.

The analytically determined test substance concentrations in the analysed test media at the start of the test varied in the range from 88 % to 96 % of the nominal values. During the test period of 72 hours a decrease of test substance concentration in the test media was determined. The mean measured test concentrations (calculated as the average over all measurements per test concentration) varied in the range of 77 % to 91 % of the nominal values. Therefore, the reported biological results are related to the mean measured test substance concentrations.

Test media of concentrations of 6.1 mg/l test item and over were slightly to strongly discoloured by the test item. Toxicity was due in part to the indirect effect of inhibited light absorption due to discolouration caused by the test item, however at higher test item concentrations a real toxic effect of the test item on the growth ofScenedesmus subspicatuscannot be excluded. The 72-hour EC50 was determined to be 14.6 mg/l based on the growth rate, and 4.8 mg/l based on biomass.

Toxicity to Micoorganisms

Toxicity of the test item to bacteria was evaluated in an experimental study according to the OECD Guideline 209 (1984). 200 ml of aerobic activated sludge, previously collected from a domestic waste water treatment plant, was combined with either the test item (at final concentrations of 3.2, 10, 32, 50 and 100 mg/l test item) or reference substance 3,5-dichlorophenol (at final concentrations of 1.0, 3.2, 10, 32 and 50 mg/l), and with bidistilled water to make 500 ml final volume. Initial pH was recorded and the flasks were kept aerated using compressed air at an air-flow rate of 0.2 l/minute. Oxygen consumption (mg O₂/l min.) was monitored for 30 minutes, and the percentage of inhibition was recorded at the end of this time.

The respiration rate of aerobic waste water bacteria was not inhibited (-2.1 % to -5.6 %) when exposed to test item concentrations between 3.2 to 100 mg/l. The EC50 (30 minutes) was therefore determined to be over 100 mg/l. The EC50 (30 minutes, ) for the reference compound, 3,5 -dichlorophenol, which was tested under the same conditions as the test item, was found to be 19.8 mg/l and therefore lies within the range of 5 - 30 mg/l, the range recommended by the test guidelines.

JUSTIFICATION FOR CLASSIFICATION  

According to the CLP Regulation (EC 1272/2008), Part 4: Environmental Hazards, the substances can be classified as hazardous to the aquatic environment when the following criteria are met:

a) A substance is classified in Category 1: Acute Aquatic Hazard when the 96 hr LC50 (fish), 48 hr EC50 (crustacea) and/or 72 or 96 hr ErC50 (algae or other aquatic plants) is ≤ 1 mg/l.

b) When adequate chronic toxicity data are not available and the substance is not rapidly biodegradable, and/or the experimentally determined BCF ≥ 500 (or, if absent, the log K_ow ≥ 4), the substance shall be classified as follows:

Category 1: Chronic Aquatic Hazard when the 96 hr LC50 (for fish), 48 hr EC 50 (crustacea) and/or 72 or 96 hr ErC50 (algae or other aquatic plants) is ≤ 1 mg/l;

Category 2: Chronic Aquatic Hazard when the 96 hr LC50 (fish), the 48 hr EC50 (crustacea) and/or 72 or 96 hr ErC50 (algae or other aquatic plants) is > 1 to ≤ 10 mg/l;

Category 3: Chronic Aquatic Hazard when the 96 hr LC50 (fish), the 48 hr EC50 (crustacea) and/or 72 or 96 hr ErC50 (algae or other aquatic plants) is > 10 to ≤ 100 mg/l.

 

The substance is not rapidly degradable and the available acute toxicity tests to fish fixed the effect levels that meet the criteria related to the hazard categories for Category Chronic 2. On the other hand invertebrates and algae fixed the effect levels that meet the criteria for Aquatic toxicity Category Chronic 3.

As fish is the most sensitive specie, the substance is classified for Aquatic toxicity as Category Chronic 2 according to the CLP Regulation (EC 1272/2008).