Disodium 3,3'-[[6-[bis(2-hydroxyethyl)amino]-1,3,5-triazine-2,4-diyl]bis[imino(3-methoxy-4,1-phenylene)azo]]bis[benzenesulphonate]

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:

July from 17 to 20, 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Version / remarks:

1984

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Details on test solutions:

Based on the results of a pre-experiment (without GLP), no stock solution could be prepared due to the limited water solubiliiy of the test item under the pre-test conditions. Therefore, 504.9 mg of the test item was directly weighed by means of an analytical balance and transferred to the test vessel with 284 ml- of tap water. The test item was mixed into the tap water by ultrasonic treatment for 15 minutes and intense stirring for three hours at room temperature in the dark, to dissolved a minimum amount of the test item and/or disperse it as homogeneously as possible. No emulsifers or solvents were used.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Laboratory culture:
- Source: ARA Ergolz II, Füllinsdorf, Switzerland.
- Pretreatment:the sludge was washed three times with tap water by centrifugation and the supernatant liquid phase was decanted.
- Method of cultivation: 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 peiod of three 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.
- Initial biomass concentration: before use, the dry weight of the activated sludge was measured again in the inoculum used for the test and diluted to 4 g dry material per liter with tap water.
- PH: the pH of the activated sludge inoculum was measured to be 6.7.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Test temperature:

20 °C

pH:

Control: 7.3 - 8.1
Test vessel: 7.4 - 7.9

Dissolved oxygen:

Control: 8.2 - 7.7 mg O2/l
Test vessel: 8.3 - 8.5 mg O2/l

Nominal and measured concentrations:

1000 mg/l, nominal

Details on test conditions:

TEST SYSTEM
- Test vessel: 2000-ml glass beakers.
- Aeration: all test media and controls were continuosly aerated by iintense stirring on magnetic stirrers to avoid possible foaming and/or stipping of the test item. The concentration of the dissolved oxygen did not drop below 2.5 mg/l during the incubation period and, just prior to measurement of the respiration rates, the dissolved oxygen were at least 8.5 mg/l.
- No. of vessels per concentration: 1 replicates.
- No. of vessels per control: 2 replicates, containing only tap water, synthetic wastewater and inoculum.
- No. of vessels per positive control: 1 replicate per concentration.
- Sludge amount: 200 ml.
- Sludge concentration: 4.0 g/l dry weight (corresponding to 1.6 g dry material per liter test medium).

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:

inhibition of total respiration

Details on results:

At the limit test concentration of 1000 mg/l, test item was not completely dissolved in the test medium. Thus, the limit test concentration was above the water solubility limit of test item under the test conditions.
The test item had no significant inhibitory effect (ca -4.5 %;) on the respiration rate of activated sludge after the incubation period of 3 hours at the limit test concentration of 1000 mg/l.
Based on the physical appearance of the test suspension, the saturation concentration (water solubility limit of the substance under the test conditions) was lower than 1000 mg/l and resulted in ca -4.5 % inhibition after 3 hours contact time. Thus, the 3-hour NOEC is at the limit of water solubility of test item under the test conditions, or at least 1000 mg/l. The 3-hour EC20, EC50 and EC80 could not be calculated, but are clearly higher than the limit of water solubility of test item under the test conditions, or 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 only by 1 % (guideline-recommended maximum variation of 15 %).

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, thus confirming suitability of the activated sludge used.

Oxygen consumption of activated sludge

Vessel	Oxygen consumption rate (mg O2/l/min)	Inhibition (%)	pH		Oxygen concentration (mg O2/l)
			Start	End	Start	End
Control	1.483	-	7.4	8.0	8.2	8.7
Control	1.463	-	7.3	8.1	8.2	8.7
Mean	1.473
Deviation (%)	1.3
Test item 1000 mg/l	1.54	-4.5	7.4	7.9	8.3	8.5
Positive control 5 mg/l	1.355	8	7.3	8.1	8.4	8.7
Positive control 16 mg/l	0.541	63.3	7.3	8.1	8.6	8.5
Positive control 50 mg/l	0.125	91.5	7.2	8.0	8.9	8.6

Conclusions:

IC50 (3h) > 1000 mg/l (nominal)
NOEC (3h) ≥ 1000 mg/l (nominal)

Executive summary:

The inhibitory effect of the test item 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.

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 significant inhibitory effect (ca -4.5 %) on the respiration rate of activated sludge after the incubation period of three hours at the limit test concentration of 1000 mg/l. Based on the physical appearance of the test suspension, the saturation concentration (water solubility limit of test item under the test conditions) was lower than 1000 mg/l and resulted in less than contact time. Thus, the 3-hour NOEC of test item to activated sludge microorganisms is at the limit of water solubility under test conditions, or at least 1000 mg/l. The 3-hour EC20, EC50 and EC80 could not be calculated, but are clearly higher than the limit of water solubility of test teim.

Conclusion

IC50 (3h) > 1000 mg/l (nominal)

NOEC (3h) ≥ 1000 mg/l (nominal)

Description of key information

No harmful/toxic to microorganisms (NOEC (3h) ≥ 1000 mg/l (nominal))

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

1 000 mg/L

Additional information

The inhibitory effect of the test item 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 209. A limit test with a nominal test concentration of 1000 mg/l was performed.

The test item had no significant inhibitory effect (ca -4.5 %) on the respiration rate of activated sludge after the incubation period of three hours at the limit test concentration of 1000 mg/l. Based on the physical appearance of the test suspension, the saturation concentration (water solubility limit of test item under the test conditions) was lower than 1000 mg/l and resulted in less than contact time. Thus, the 3-hour NOEC of test item to activated sludge microorganisms is at the limit of water solubility under test conditions, or at least 1000 mg/l.