trisodium 3-[(E)-2-(4-{[4-chloro-6-({2-[(4-{[4-(ethenesulfonyl)phenyl]amino}-6-fluoro-1,3,5-triazin-2-yl)amino]propyl}amino)-1,3,5-triazin-2-yl]amino}-5-sulfonaphthalen-1-yl)diazen-1-yl]naphthalene-1,5-disulfonate

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:

04 September 2006 to 17 November 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:

not specified

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identity: FAT 40826/A
Batch no.: TZ 5604 BOP 01/06
Expiration date: February 01, 2011
Purity: Content of organic part (Na-salt): approx. 78 %; Oligomers: 13 %; Main component: approx. 48 %
Solubility in water: Approx. >50 g/L at room temperature
Stability in water: Max. 7 days at room temperature
pH: 7.6 (1 g/L)
Aggregate state/physical form at room temperature: Solid (orange powder)
Storage conditions: At room temperature at about 20 °C, away from direct sunlight
Specific instructions: Store in desiccator

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

Based on the results of a pre-experiment (without GLP), the test item was soluble at a concentration of at least 5 g/L in tap water. Therefore, 500.58 mg of the test item was directly dissolved in 284 ml tap water by ultrasonic treatment for fifteen minutes and intense stirring for ten minutes at room temperature. Then, 16 ml synthetic wastewater and 200 ml activated sludge inoculum were added. After the stirring period, 16 mL of synthetic wastewater and 200 mL of the activated sludge inoculum were added.

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, FüIIinsdorf, Switzerland) treating predominantly domestic wastewater. To eliminate possible inhibitory material, 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 adjusted from 8.1 to 7.2 with a diluted sulfuric acid solution.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Test temperature:

19 °C

pH:

7.7-8.3

Dissolved oxygen:

8.0-8.5 mg/L

Nominal and measured concentrations:

The final nominal concentration is 1000 mg/L

Details on test conditions:

The test was performed in 2000-ml glass beakers. The test vessels were labeled with the necessary information to ensure unmistakable identification. At the start of the test (after the stirring period of ten minutes), synthetic wastewater and activated sludge inoculum were added. The inoculum had a sludge concentration of 2.2 g/L dry weight (corresponding to about 0.9 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 three hours, the single test medium 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. Just before measurement of the respiration rates, the dissolved oxygen concentrations were at least 8.2 mg/L. The temperature in the test media. measured in one control, was 20 °C at the start and at the end of the incubation period.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Duration:

3 h

Dose descriptor:

IC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth inhibition

Details on results:

The test item FAT 40826/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 (EC15) of FAT 40826/A to activated sludge microorganisms was at least 1000 mg/L This value might even be higher, but concentrations above 1000 mg/L were not tested. The 3-hour EC20, EC50, and EC80 could not be calculated but were clearly higher than 1000 mg/L.

Results with reference substance (positive control):

The 3-hour EC50 of the reference item 3,5-dichlorophenol (positive control) was calculated to be 14 mg/L (the 95 % confidence limits were not calculable). The 3-hour EC50 is within the guideline-recommended range of 5-30 mg/L, confirming suitability of the activated sludge used.

Validity criteria fulfilled:

yes

Conclusions:

The 3-h EC20, EC50 and EC80 of FAT 40826/A to activated sludge microorganisms was clearly higher than 1000 mg/L.

Executive summary:

In a GLP-compliant study, the inhibitory effect of the test substance on the respiration rate of aerobic waste water microorganisms of activated sludge was investigated in a 3-hour respiration inhibition test according to the EU method C.11 and the OECD Guideline 209. 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 had no inhibitory effect on the respiration rate of activated sludge after the incubation period of three hours at the test item concentration of 1000 mg/L. Thus, the 3-hour NOEC (EC15) of the test item to activated sludge microorganisms was at least 1000 mg/L. This value might even be higher but concentrations above 1000 mg/L were not tested. Based on the test results, the 3-h EC20, EC50, and EC80 could not be calculated, but were clearly higher than 1000 mg/L.

Description of key information

The 3-h EC50 value of test substance is >1000 mg/L in microorganisms.	

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L

EC10 or NOEC for microorganisms:

1 000 mg/L

Additional information

In a GLP-compliant study, the inhibitory effect of the test substance on the respiration rate of aerobic waste water microorganisms of activated sludge was investigated in a 3-hour respiration inhibition test according to the EU method C.11 and the OECD Guideline 209. 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 had no inhibitory effect on the respiration rate of activated sludge after the incubation period of three hours at the test item concentration of 1000 mg/L. Thus, the 3-hour NOEC (EC15) of the test item to activated sludge microorganisms was at least 1000 mg/L. This value might even be higher but concentrations above 1000 mg/L were not tested. Based on the test results, the 3-h EC20, EC5O, and EC80 could not be calculated, but were clearly higher than 1000 mg/L.