Salts of 9-[2-(ethoxycarbonyl)phenyl]-3,6-bis(ethylamino)-2,7-dimethylxanthenium chloride and 4-{[1-hydroxy-6-({[5-hydroxy-6-(phenyldiazo)-7-sulfonaphthalen-2-yl]carbamoyl}amino)-3-sulfonaphthalen-2-yl]diazo}benzoic acid (2:1)

Endpoint summary

Additional information

Fish

Young fish were exposed to aqueous test media containing the test item at the nominal concentrations: 5.535; 6.912; 8.64; 10.8; and 13.5 mg/L. For the analysis of the test item concentrations, samples from the treatment groups and the untreated control were taken at the start and end of the study. The analytically determined mean test item concentrations in the analysed test media ranged from 87 % to 92 % at the start and from 54 % to 67 % at the end of the study. The deviation of the measured concentrations from the nominal values was higher than 20 % at all concentration at the end of the study. Therefore, all reported biological results are related to the geometric mean measured concentrations of the test item calculated by EXCEL Software Program. The corresponding measured mean concentrations were 3.84; 4.79; 6.53; 8.49 and 10.62 mg test item/L. The 96-hour LC50 value was calculated to be 12.18 mg/L (Probit analysis), the 96-hour LC0 and the overall NOEC were determined to be 6.53 mg/L. The 96-hour LC100 could not be determined because there was no 100% mortality during the test. As the deviation of the measured concentrations from the nominal values was higher than 20 %, all reported biological results are related to measured concentrations. In this 96-hour static acute toxicity test to Zebrafish (Brachydanio rerio) with F 213 Red the 96h LC50 value was 12.18 mg/L (Probit analysis) based on measured concentrations. The overall NOEC was determined to be 6.53 mg/L, based on measured concentrations.

Daphnia

The analytically determined mean test item concentrations in the analysed test media varied in the range from 102 % to 119 % at the start and from 52 % to 97 % at the end of the study in comparison to the nominal value. The 48h-NOEC (highest concentration tested without toxic effects after the exposure period of 48 hours), respectively the 48h-EC0 of F 213 Red to Daphnia magna was determined to be 0.012 mg test item/L. The 48h-EC50 was calculated to be 0.030 mg test item/L and the 48h-EC100 was determined to be 0.103 mg test item/L In this 48-hour static acute toxicity test to Daphnia magna with F 213 Red the 48 h EC50 value was 0.030 mg/L (Probit analysis), based on measured concentrations. The overall NOEC was determined to be 0.012 mg/L, based on measured concentrations.

Algal Growth Inhibition

The algal growth in relation to a control culture was determined over a fixed test period of 72 hours and thus, over several algal generations. The test method of application and the test species Pseudokirchneriella subcapitata are recommended by the test guidelines. Nominal test item concentrations: 0.008; 0.016; 0.031; 0.063; 0.125 and 0.25 mg/L Measured test item concentrations (mean): 0.004; 0.006; 0.012; 0.017; 0.024 and 0.052 mg/L The test item concentration of the test solutions was measured by HPLC-UV method at the start and at the end of the test. The analytically determined test item concentrations in the analysed test media varied in the range from 65% - 91% at the start and from 5% - 27% at the end of the study in comparison to the nominal value (see the attached analytical report). The deviation of the measured concentrations from the nominal values was higher than 20 %. Therefore, all reported biological results are related to the geometric mean of the measured test item concentrations at the start and at the end of the test calculated by EXCEL Software Program. F 213 Red had a statistically significant inhibitory effect on the growth based on the average specific growth rate, yield and areas under the growth curves of Pseudokirchneriella subcapitata after the exposure period of 72 hours in the concentration range of 0.0168 – 0.0522 mg test item/L (based on the measured mean concentrations). The test concentration of 0.0168 mg/L (measured mean) was determined as the 72-hour LOEC. The test concentration of 0.0124 mg/L (measured mean) was determined as the 72-hour NOEC. The 72-h EC50 based on growth rate was determined as 0.143 mg/L, the 72-h EC50 based on yield 0.046 mg/L. The overall NOEC was determined to be 0.012 mg/L (all based on the measured mean concentrations). The nominal test item concentrations were 0.008; 0.016; 0.031; 0.063; 0.125 and 0.25 mg test item/L. The measured test item concentrations were out the ±20 % range of the nominal concentrations at the start (65 % - 91 %) and at the end (5 % - 27 %) of the test. Therefore, all reported biological results are related to the geometric mean of the measured test item concentrations at the start and at the end of the test calculated by EXCEL Software Program. The corresponding measured mean test item concentrations were: 0.004; 0.006; 0.012; 0.017; 0.024 and 0.052 mg/L Growth Inhibition: The test item had a statistically significant inhibitory effect on the growth based on the average specific growth rate, yield and areas under the growth curves of Pseudokirchneriella subcapitata after the exposure period of 72 hours in the concentration range of 0.0168 – 0.0522 mg test item/L (based on the measured mean concentrations). The test concentration of 0.0168 mg/L (measured mean) was determined as the 72-hour LOEC. The test concentration of 0.0124 mg/L (measured mean) was determined as the 72-hour NOEC.

Activated Sludge

In comparison to the inoculum controls the respiration rate of the activated sludge was not inhibited in the concentration range of 10 to 1000 mg F 213 Red /L. The respiration rate was inhibited between 1.9 % (10 mg F 213 Red/ L) and 13.0 % (1000 mg F 213 Red/ L) in the examined nominal test concentration range. Concentrations exceeding 1000 mg/L nominal were not tested. The NOEC (3h) and EC50 (3h) were both determined to be > 1000 mg/l.

The acute aquatic toxicity tests for F 213 Red revealed an LC50 (96 h) of 12.18 mg/L in fish, an EC50 (48 h) of 0.03 mg/L in invertebrates and an EC50 (72 h, growth rate) of 0.143 mg/L. The most sensitive test for the aquatic compartment was the short-term toxicity to aquatic invertebrates, with an EC50 (48h) of 0.03 mg/L. This value was used for the calculation of the predicted no effect concentration.

Based on the acute aquatic toxicity results obtained, F 213 Red is classified and labelled N; R50/53 (67/548/EEC) and H410 (M10), Chronic Cat. 1, Warning (CLP).