Potassium sodium 4,4'-bis[6-anilino-4-[bis(2-hydroxyethyl)amino]-1,3,5-triazin-2-yl]amino]stilbene-2,2'-disulphonate

Long-term toxicity to fish

Description of key information

NOEC (fry length) < 0.625 mg/l (nominal)

NOEC (fry weight) = 0.625 mg/l (nominal)

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

0.625 mg/L

Additional information

The effects of the test item on the early-life stage of fish were evaluated by considering data on two similar substances 3a-A(Na), 2 -A and 5 -A, in a weight of evidence approach. Justification for Read Across is given in Section 13 of IUCLID.

The study on 2 -A was performed according to OECD Guideline 210. Based on the results of a range finding test the test was conducted as a dose-response test with the nominal test item concentrations 0.625, 1.25, 2.50, 5.00 and 10.0 mg/l. The test was started by placing fertilized eggs into the test vessels and it lasted 35 days (30 days post-hatch). 80 eggs of Danio rerio / zebrafish were exposed to each test concentration, the solvent control and the control (4 replicates with 20 eggs each). On study day 5, 91% of the control and 90% the solvent control larvae had hatched. Therefore, study day 5 was defined as post hatch day 0 (= PHD 0). Different toxicological endpoints were determined: hatching success, fry growth (assessed via length and fresh weight measurements on PHD 30), morphological and behavioral effects, posthatch survival and overall survival. Specific analysis of various concentrations of the substance the test media and the controls was carried out via HPLC-DAD. The measured concentrations of replicates and test media on study days 0 to 28 were in the range of 81 to 104% of the nominal concentrations. All effect values are given based on the nominalconcentrations of the test item. The results of the parameters hatching success, fry growth (expressed as weight and length measurement at PHD 30), post-hatch survival and overall survival were checked for statistically significant differences. No statistically significant difference was detected between the dilution water control and the solvent control for all parameters (hatching success, fry growth expressed as length or weight on PHD 30, post-hatch survival and overall survival). Therefore, both controls were pooled for statistical analysis.

The test item caused significant effects on Zebrafish in an early life stage test, 30 days post hatch. For the parameter hatch, the NOEC was 10.0 mg/l. For the parameters post hatch survival and overall survival, the NOECs were 1.25 and 2.50 mg/l, respectively. For the parameter fry growth (expressed as length and fresh weight) the NOECs were < 0.625 mg/l (length) and 0.625 mg/l (weight).

The effects of the similar substance 3a-A(Na) on the early-life stage of fish (Danio rerio/ Zebrafish) were determined according to OECD Guideline 210. Methanol was used as solvent with a concentration of 0.10 ml/l dilution water. Based on the results of a range finding test the test was conducted as an extended Limit test with the nominal test item concentrations of 1.00 and 10.0 mg/l. The test was started by placing fertilized eggs into the test vessels and it lasted 35 days (30 days post-hatch). 80 eggs of Danio rerio / zebrafish were exposed to each test concentration, the solvent control and the control (4 replicates with 20 eggs each). The water quality parameters pH-value, oxygen concentration, temperature and total hardness were within the acceptable limits.

On study day 5, 95% of the control and 96 % the solvent control larvae had hatched. Therefore, study day 5 was defined as post hatch day 0 (= PHD 0). Different toxicological endpoints were determined: hatching success, fry growth (assessed via length and fresh weight measurements on PHD 30), morphological and behavioral effects, posthatch survival and overall survival. Specific analysis of various concentrations of the substance in the test media and the controls was carried out via HPLC-DAD. The test media were sampled and analyzed prior to exposure on days -1 and during the exposure on study days 0, 2, 7, 8, 9, 14, 21, 22, 28 and 29. The measured concentrations of replicates and test media on study days 0 to 35 were in the range of 109 to 119% of the nominal concentrations in the nominal test item concentration 10.0 mg/l and in the range of 26 to 136% of the nominal concentrations in the nominal test item concentration 1.00 mg/l. Since the measured concentrations were partly out of the range of ± 20% of the nominal concentration, the time-weighted arithmetic mean measured concentrations of the test item were calculated, resulting in 1.09 and 11.2 mg/l, respectively. The effect values NOEC, LOEC, ECx, LCx values were determined based on the statistical results.

The test item partly caused significant effects on Zebrafish in an early life stage test, 30 days post hatch when tested with time-weighted arithmetic mean measured concentrations of 1.09 and 11.2 mg/l. For the parameters hatch, post-hatch survival, overall survival and fry growth (expressed as length) the NOEC was 11.2 mg/l. For the parameter fry growth (expressed as fresh weight) the NOEC was < 1.09 mg/l.

The effects of the test item on the early-life stage of fish (Danio rerio / Zebrafish) on the substance 5 -A were determined at the test facility according to OECD Guideline 210.DMSO was used as solvent with a concentration of 0.10 mL/l dilution water. Stock solutions in DMSO with nominal concentrations of 125 and 12.5 g/l were prepared in appropriate intervals of 4 to 7 days and continuously dosed to the dilution water in a flow-through system. Based on the results of a range finding test the test was conducted as an extended Limit test with the nominal test item concentrations 1.25 and 12.5 mg/l. The test was started by placing fertilized eggs into the test vessels and it lasted 36 days (30 days post-hatch). 80 eggs of Danio rerio / zebrafish were exposed to each test concentration, the solvent control and the control (4 replicates with 20 eggs each). The water quality parameters pH-value, oxygen concentration, temperature and total hardness were within the acceptable limits. On study day 6, 90% of the control and 89% of the solvent control larvae had hatched. Therefore, study day 6 was defined as post hatch day 0 (= PHD 0). Different toxicological endpoints were determined: hatching success, fry growth (assessed via length and fresh weight measurements on PHD 30), morphological and behavioral effects, posthatch survival and overall survival.

Specific analysis of various concentrations of the test item in the test media and the controls was carried out via HPLC-DAD. The test media were sampled and analyzed prior to exposure on days -1 and during the exposure on study days 0, 6, 13, 20 and 30. The measured concentrations of replicates and test media on study days 0 to 35 were in the range of 94 to 108% of the nominal concentrations in the nominal test item concentration 12.5 mg/l and 1.25 mg/l. Since the measured concentrations were in the range of ± 20% of the nominal concentration, the nominal concentrations of the test item were used for the evaluation. The DMSO stock solutions were sampled and analyzed from freshly prepared and corresponding 7 days aged stock solution.

All effect values are given based on the nominal concentrations of the test item. The results of the parameters hatching success, fry growth (expressed as weight and length measurement at PHD 30), post-hatch survival and overall survival were checked for statistically significant differences. No statistically significant difference was detected between the dilution water control and the solvent control for all parameters (hatching success, fry growth expressed as length or weight on PHD 30, post-hatch survival and overall survival). Therefore, both controls were pooled for statistical analysis. The effect values NOEC, LOEC, ECx, LCx values were determined based on the statistical results.

The test item partly caused significant effects on Zebrafish in an early life stage test, 30 days post hatch when tested with nominal concentrations of 1.25 and 12.5 mg/l. For the parameters hatch, post-hatch survival and overall survival the NOEC was 12.5 mg/l. Therefore, the respective LOECs were determined to be > 12.5 mg/l. For the parameters fry growth (expressed as fresh weight and length) the NOEC was < 1.25 mg/l. Therefore, the LOEC for these parameters was determined to be 1.25 mg/l. All effect values are given based on the nominal concentrations of the test item.

The study conducted on the Similar substance 2 -A is used for the chemical safety assessment, following a conservative approach. More information on the results of the substances of the category are found on the document attached under "Ecotoxicological information"