[No public or meaningful name is available]

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

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)

Vehicle:

no

Details on test solutions:

The test item was mixed into the tap water using ultrasonic treatment over 15 minutes and intense stirring over 24 hours at room temperature in the dark to dissolve a maximum amount of the test item and/or disperse it as homogeneously as possible. No emulsifiers or solvents were used.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Source: ARA Ergolz II, Füllinsdorf, Switzerland.
- Pre-treatment: 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.
- Feed: the sludge was fed daily with 50 ml synthetic wastewater per liter.
- Storage conditions: sludge was kept at room temperature under continuous aeration until use.
- Preparation of inoculum for exposure: an aliquot of washed sludge was suspended in tap water to obtain a concentration equivalent to 3 g dry material per liter.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

20 °C

Nominal and measured concentrations:

10, 32, 100, 320 and 1000 mg/l

Details on test conditions:

TEST SYSTEM
- Test vessel: 2000-ml glass beakers.
- Aeration: during the incubation period all test media and the controls were continuously aerated by intense stirring on magnetic stirrers.
- No. of vessels per concentration: 1 replicate.
- No. of vessels per control: 2 replicate.
- Sludge amount: 200 ml
- Slude goncentration: concentration of 4 g/l dry weight, which corresponds to about 1.6 g dry material per liter test medium.

TEST MEDIUM / WATER PARAMETERS
- Synthetic wastewater: 16 ml of synthetic wastewater.

EFFECT PARAMETERS MEASURED
Then the dissolved oxygen concentration was measured with an oxygen electrode and was continuously recorded for about ten to fifteen minutes. During measurement, the samples were continuously stirred on a magnetic stirrer. The oxygen consumption rate was determined from the linear part of the respiration curve in the range of 6.5-2.5 mg O2/l.

TEST CONCENTRATIONS
- Test concentrations: 1000 mg/l
- Results used to determine the conditions for the definitive study: an inhibition on the respiration rate of activated sludge of 22 % was observed.

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

Details on results:

Up to and including the concentration of 320 mg/l the test item had no significant inhibitory effect (< 15 %) on the respiration rate of activated sludge after the incubation period of 3 hours. At the highest test item concentration of 1000 mg/l the inhibitory effect was 29 %.

CONTROL
The oxygen consumption rates of the two controls (run at the start or at the end of the test) differed only by 1 %.

Results with reference substance (positive control):

The 3-hour EC50 of the reference item 3,5-dichlorophenol (positive control) was 17 mg/l. The 3-hour EC50 is within the guidelinere commended range of 5-30 mg/l, confirming the suitability of the activated sludge used.

Table 1: Results.

Test chemical (mg/l)	Oxygen consumption rate (mg O2/l/min)	Inhibition (%)	pH values		Oxygen concentration (mg O2/l)
			Start*	End*	Start*	End*
Control	1.100	-	7.4	8.4	8.4	8.3
Control	1.085	-	7.9	8.4	8.1	8.3
Mean	1.093 (deviation: 1.3 %)
Test item 10	1.148	-5.1	8.0	8.5	7.9	8.3
Test item 32	1.166	-6.7	7.9	8.5	7.8	8.9
Test item 100	1.140	-4.3	7.9	8.4	8.0	8.9
Test item 320	1.045	4.4	8.0	8.5	8.0	9.0
Test item 1000	0.780	28.6	8.1	8.5	8.0	8.4
3,5-dichlorophenol 5	1.040	1.8	7.8	8.5	8.1	8.4
3,5-dichlorophenol 16	0.542	50.4	7.9	8.5	8.4	8.0
3,5-dichlorophenol 50	0.119	89.1	7.9	8.6	8.3	8.6

*Start and end of the 3-hour incubation period

- % inhibition: increased oxygen consumptionrate relative to that of control

Validity criteria fulfilled:

yes

Conclusions:

EC50 (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 209. The following nominal test item concentrations were tested: 10, 32, 100, 320 and 1000 mg/l. Concentrations exceeding 1000 mg/l were not tested. In addition, two controls and three different concentrations of a reference item, 3,5 -dichlorophenol (5, 16 and 50 mg/l), were tested in parallel. The results of these treatments confirmed the suitability of the activated sludge and the method used.

Up to and including the concentration of 320 mg/l, the test item had no significant inhibitory effect (< 15 %) on the respiration rate of activated sludge after an incubation period of 3 hours. At the highest test item concentration of 1000 mg/l, the inhibitory effect was 29 %.

The calculated 3-hour EC-values were as follows: NOEC (determined as the calculated 3-hour EC₁₅) = 631 mg/l; EC₅₀ > 1000 mg/l. The 95% confidence limits could not be determined.

Conclusion

EC₅₀ (3h) >1000 mg/l (nominal)

Description of key information

EC₅₀ (3h, aerobic wastewater microorganisms) > 1000 mg/l (nominal)

EC₁₅ (3h) = NOEC (3h) = 631 mg/l (nominal)

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 EU Commission Directive 88/302/EEC, Part C.11, and the OECD guideline 209. The following nominal test item concentrations were tested: 10, 32, 100, 320 and 1000 mg/l. Up to and including the concentration of 320 mg/l, the test item had no significant inhibitory effect (< 15 %) on the respiration rate of activated sludge after an incubation period of 3 hours. At the highest test item concentration of 1000 mg/l, the inhibitory effect was 29 %.

Additionally, an experimental study on a structural analogue demonstrated that the EC50 value of Similar Substance 01 is also greater than 1000 mg/l (OECD 209). This data supports the findings of the experimental study on Direct Blue 267.