2,5-cyclohexadien-1-one, 4-[[4-(phenylamino)phenyl]imino]-, reaction products with sodium sulfide (Na2(Sx)), leuco derivatives

Toxicity to microorganisms

Description of key information

In an Activated Sludge Respiration Inhibition Test with the read across substance Blue Sema (CAS No 1040873-93-5), the EC10 and EC50 values were determined as greater than 1000 mg/L. Based on the statistical evaluation in this test the NOEC was 1000 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

1 000 mg/L

Additional information

No data on toxicity to aquatic microorganisms were available for the registration substance Blue 13 (CAS No 12262-26-9) itself. Therefore, a read across to the structural analogue Blue Sema (CAS No 1040873-93-5) was conducted.

To evaluate the influence of the structural analogue Blue Sema on the activity of activated sludge, a 3-hour Activated Sludge Respiration Inhibition Test according to OECD Guideline 209, EU Method C.11 and US EPA OCSPP 850.3300 was conducted. In a preliminary test, the respiration rates (total, heterotrophic and nitrification oxygen uptake rates) of samples of activated sludge fed with synthetic sewage were measured in an enclosed cell containing an oxygen electrode after a contact time of 3 hours. The structural analogue was investigated in this study at the nominal concentrations of 10, 100 and 1000 mg/L. Defined amounts of the ground test item were added directly into the test vessels. Triplicate vessels were prepared and investigated at the highest examined test item concentration. In parallel with the test item treatments 3,5-Dichlorophenol as positive reference control in a concentrations of 2, 7 and 24.5 mg/L; furthermore, blank (inoculum) control, nitrification controls and abiotic controls were investigated. Abiotic controls (investigated in three parallels) were prepared containing the test item in the concentration of 1000 mg/L, synthetics sewage feed, but no inoculum. In this test no abiotic oxygen consumption was noticed. The test was performed without pH adjustment. During the test, significant effect of the test item on the pH was realized at the highest examined concentration of 1000 mg/L. The pH of the test mixtures in presence of 1000 mg/L (the highest examined concentration) test item was ~5 without inoculum, but within the 7—8 pH range after inoculation.The test item may adversely affect pH within the test system; however the results of the present preliminary test show, that the slightly lower pH caused by the test item has no effect on the applied inoculum culture and further testing (a definite test) with additional neutralization step of test item containing mixtures before inoculum addition is not necessary. All validity criteria of the study were met. The average specific respiration rate of the blank was 33.69 mg O2/ g activated sludge (based on dry weight) in an hour with a coefficient of variation of 2.87 %. The 3-hour EC50 of the reference item 3,5-Dichlorophenol was 13.87 mg/L within the range of 2 mg/L to 25 mg/L, that was required for total respiration (in this study thedifferentiation between heterotrophic respiration and nitrification was considered as not necessary). The observed oxygen consumption rates and consequently the specific respiration rates in all examined test item concentrations remained in the range of the blank controls (the average specific respiration rate at 1000 mg/L: 32.87 mg O2/gh). No inhibitory effect of the test item was observed.

Based on measured oxygen consumption values and calculated specific respiration rates it can be statedthat the 3-hour EC10and EC50 values of the test item are greater than 1000 mg/L. The NOEC was determined to be 1000 mg/L, the highest concentration tested. The specific respiration rates at the examined highest test item concentration of 1000 mg/L were compared with the blank controlvalues using 2-Sample T-test (a=0.05). No statistical significant differences were observed, compared tothe blank control values. In conclusion, this preliminary test demonstrated the absence of inhibition of oxygen consumption by the test substance up to and including the limit concentration of 1000 mg/L, therefore in line with OECD 209 a definite test is not required.

Based on the results of the preliminary test with the structural analogue Blue Sema (CAS No 1040873-93-5), the EC50 and EC10 of the registration substance Blue 13 (CAS No 12262-26-9) was determined to be > 100 g/L and the NOEC to be 1000 mg/L.