Tetrasodium [2-({4-fluoro-6-[(2-{[4-fluoro-6-({5-(hydroxykappaO)-6-[(2-{[2-(hydroxy-kappaO)-5-sulfophenyl] diazenyl-kappaN1}-4,5-dimethoxyphenyl) diazenyl-kappaN2]-7-sulfo-2-naphthyl} amino)-1,3,5-triazin-2-yl] amino} propyl) amino]-1,3,5-triazin-2-yl} amino) benzene-1,4- disulfonato(6-)] cuprate(4-)

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental Starting Date - 12 June 2006;
Experimental Completion Date - 14 June 2006;
Study completion date - 08 September 2006.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identity: FAT 40825/A
Batch number: CHU 297 / BOP 04/05
Purity: Organic part (Na-salt): approx. 83.7 %; All coloured components: approx. 80.54 %; Main component: approx. 59.1 %.
Appearance: Solid, black powder
Storage conditions: At room temperature at about 20 °C
Expiration date: December 31, 2010

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

According to the results of a pre-experiment (without GLP), the test item is soluble at a concentration of at least 5 g/L in tap water. For the limit concentration of 1000 mg/L to be tested, 500.7 mg of the test item was dissolved in 284 mL tap water by intense stirring at room temperature for fifteen minutes. Then, 16 mL synthetic wastewater and 200 mL activated sludge inoculum were added. Additionally, two controls containing only tap water, synthetic wastewater and inoculum were tested in parallel to the limit test concentration under identical test conditions.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

The study was performed with aerobic activated sludge from a wastewater treatment plant (ARA Ergolz II, Füllinsdorf, Switzerland) treating predominantly domestic wastewater. The sludge was washed twice with tap water by centrifugation and the supernatant liquid phase was decanted. A homogenized aliquot of the final sludge suspension was weighed, thereafter dried and the ratio of wet to dry weight was calculated. Based on this ratio, an aliquot of washed sludge was suspended in tap water to obtain a concentration equivalent to 3 g dry material per liter. During the holding period of two days prior to use, the sludge was fed daily with 50 mL synthetic wastewater* per liter and was kept at room temperature under continuous aeration until use. Before use, the dry weight of the activated sludge was measured again in the inoculum used for the test. The pH of the activated sludge inoculum was measured to be 6.5.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Test temperature:

20 - 21 °C

pH:

7.3 - 8.3

Nominal and measured concentrations:

nominal: 1000 mg/L

Details on test conditions:

Experimental Conditions
The test was performed in 2000-mL glass beakers. The test vessels were labelled with the necessary information to ensure unmistakable identification. At the start of the test (after dissolution), synthetic wastewater and activated sludge inoculum were added. The inoculum had a sludge concentration of 2.7 g/L dry weight (corresponding to 1.1 g dry material per liter test medium). The sludge was added in time intervals of 15 minutes (an arbitrary but convenient interval) first to a control, secondly to the test solutions of the reference item, thirdly to the test solution of the test item, and finally to the second control. During the incubation period of exactly 3 hours, all test media and the controls were continuously aerated by intense stirring on magnetic stirrers to avoid possible foaming and/or stripping of the test item. The concentration of dissolved oxygen did not drop below 2.5 mg/L during the incubation period, and just before measurement of the respiration rates, the dissolved oxygen concentrations were at least 8.4 mg/L. The temperature in the test media, measured in one control, was 20 °C at the start and 21 °C at the end of the incubation period.

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

>= 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Details on results:

At the limit test concentration of 1000 mg/L, FAT 40825/A was completely dissolved. The test item FAT 40825/A had no significant inhibitory effect (<15%) on the respiration rate of activated sludge after the incubation period of three hours at the limit test concentration of 1000 mg/L. Thus, the 3-hour NOEC of FAT 40825/A to activated sludge microorganisms is at least 1000 mg/L. The 3-hour EC20, EC50 and EC80 could not be calculated, but are clearly higher than 1000 mg/L. The oxygen consumption rates of the two controls (run at the start and at the end of the test) differed by 14% (guideline-recommended maximum variation: 15 %)

Results with reference substance (positive control):

The 3-hour EC50 of the reference item 3,5-dichlorophenol (positive control) was calculated to be 11 mg/L (the 95 % confidence limits were not calculable). The 3-hour EC50 is within the guideline-recommended range of 5 - 30 mg/L, thus confirming suitability of the activated sludge used.

Reported statistics and error estimates:

The 3-hour EC20, EC50 and EC80 of the test item could not be calculated because of the absence of an inhibitory effect on activated sludge microorganisms. The 3-hour EC50 of the reference item 3,5-dichlorophenol was calculated by Probit Analysis.

Validity criteria fulfilled:

yes

Conclusions:

The 3-hour EC50 was higher than 1000 mg/L. the NOEC was set to 1000 mg/L.

Executive summary:

The inhibitory effect of the test item FAT 40825/A 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, and the OECD Guideline for Testing of Chemicals, No. 209 in a GLP-certified laboratory. In accordance with the EU Commission Directive 92/69/EEC (C.1, C.2, and C.3), a limit test with a nominal test concentration of 1000 mg/L was performed to demonstrate that the test item has no toxic effect on activated sludge microorganisms up to and including this concentration. 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. The test item FAT 40825/A had no significant inhibitory effect (< 15 %) on the respiration rate of activated sludge after the incubation period of three hours at the limit test concentration of 1000 mg/L. Thus, the 3-hour NOEC of FAT 40825/A to activated sludge microorganisms is at least 1000 mg/L. The 3-hour EC20, EC50 and EC80 could not be calculated, but are clearly higher than 1000 mg/L.

Description of key information

The 3-hour NOEC of FAT 40825/A to activated sludge microorganisms is at least 1000 mg/L. The 3-hour EC20, EC50 and EC80 could not be calculated, but are clearly higher than 1000 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L

EC10 or NOEC for microorganisms:

1 000 mg/L

Additional information

The inhibitory effect of the test item FAT 40825/A on the respiration rate of aerobic wastewater microorganisms of activated sludge was investigated in a 3-hour respiration inhibition test according to the OECD Guideline for Testing of Chemicals, No. 209. A limit test with a nominal test concentration of 1000 mg/L was performed to demonstrate that the test item has no toxic effect on activated sludge microorganisms up to and including this concentration. 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. The test item FAT 40825/A had no significant inhibitory effect (< 15 %) on the respiration rate of activated sludge after the incubation period of three hours at the limit test concentration of 1000 mg/L. Thus, the 3-hour NOEC of FAT 40825/A to activated sludge microorganisms is at least 1000 mg/L. The 3-hour EC20, EC50 and EC80 could not be calculated, but are clearly higher than 1000 mg/L.