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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 01-Sept-2008 to 04-Sept-2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
This study was performed according to OECD Guideline 209 and EU Method C.11 with GLP statement. All validity criteria were fulfilled. The results are based on nominal concentration.
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
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Remarks:
2008-06-09
Specific details on test material used for the study:
- Storage condition of test material: in a refrigerator at about 4°C.
- Water solubility: 1.88 mg/L
Analytical monitoring:
no
Details on sampling:
Not applicable
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances):
See table 1 in "Any other information on materials and methods incl. tables".
- Method: 50.6 mg of the test item was weighed by means of an analytical balance and transferred to the designated test vessel with 284 mL tap water. The test item was mixed into the tap water by ultrasonic treatment for fifteen minutes and intense stirring for 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.
After stirring, the appearance of the test medium was evaluated, and the test concentration was clearly above the water solubility limit of the test item under the test conditions. Then, 16 mL synthetic wastewater and 200 mL activated sludge inoculum were added.
- Eluate: tap water
- Differential loading: no data
- Controls: two controls containing only tap water, synthetic wastewater and inoculum were tested in parallel to the single test concentration of the test item under identical test conditions.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): not applicable
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): not applicable
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 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. 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 8.3 to 7.0 with a diluted sulfuric acid solution.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Remarks on exposure duration:
none
Post exposure observation period:
none
Hardness:
No data
Test temperature:
The temperature in the test media, measured in one control, was 21°C at the start and at the end of the incubation period.
pH:
See table 2 in "Any other information on results incl. tables"
In the control: varied between 7.4 to 8.2 during the 3-hour incubation period.
In the test item: varied between 7.7 to 8.0 during the 3-hour incubation period.
In the reference item: varied between 7.5 to 8.2 during the 3-hour incubation period.
Dissolved oxygen:
See table 2 in "Any other information on results incl. tables"
In the control: varied between 7.9 to 8.4 mg O2/L during the 3-hour incubation period.
In the test item: varied between 8.3 to 8.4 mg O2/L during the 3-hour incubation period.
In the reference item: varied between 8.1 to 8.4 mg O2/L during the 3-hour incubation period.
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal concentraion: loading rate of 100 mg/L.
Measured concentrations:
Details on test conditions:
TEST SYSTEM
- Test vessel: glass beakers
- Material, size, headspace, fill volume: 2000 mL glass beakers
- Aeration: During the incubation period of 3 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.
- Type of flow-through (e.g. peristaltic or proportional diluter): not applicable
- Renewal rate of test solution (frequency/flow rate): not applicable
- No. of organisms per vessel: not applicable - activated sludge per vessel = 200 mL
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 2
- No. of vessels per vehicle control (replicates): not applicable
- Biomass loading rate: 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.

TEST MEDIUM / WATER PARAMETERS
no data

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: no data
- Light intensity: no data

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The respiration rates (oxygen consumption) of activated sludge were measured. The respiration rate in the presence of the test item was compared to the mean respiration rate of two controls.
For measurement of the respiration rate a well-mixed sample of each test medium was poured into a BOD-flask after three hours incubation time, and was not further aerated. Then the dissolved oxygen concentration was measured with an oxygen electrode (WTW TriOxmatic® 300 and an oxygen meter WTW Oxi 539, Wissenschaftlich-Technische Werkstaetten WTW, Weilheim/Germany), and was continuously recorded. During measurement, the samples were continuously stirred on a magnetic stirrer. The oxygen consumption rate (in mg O2/L/minute) was determined from the linear part of the respiration curve in the range 6.5–2.5 mg O2/L. In case of very rapid oxygen consumption, the range used was below the limits indicated above but always within the linear part of the respiration curve. In case of low oxygen consumption the rate was determined over a period of at least ten minutes.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: not applicable (limit test)
- Justification for using less concentrations than requested by guideline: not applicable
- Range finding study: no. A limit test was performed in accordance with the EU Commission Regulation 440/2008 (C.1-C.3) to demonstrate that the test item has no toxic effect on activated sludge up to the nominal test concentration of 100 mg/L.
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: >= limit of solubility
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: > limit of solubility
Details on results:
See table 2 in "Any other information on results incl. tables" and Figure in "Attached background material".

At the test item concentration of 100 mg/L, the test item was not completely dissolved in the test medium. Thus, the test concentration was clearly above the water solubility limit of the test item under the test conditions.

The test item had no significant inhibitory effect on the respiration rate of activated sludge after the incubation period of 3 hours at the limit test concentration of 100 mg/L.

Based on the physical appearance of the test suspension, the saturation concentration (water solubility limit of the test item under the test conditions) was <100 mg/L and resulted in 1.4% inhibition after three hours contact time.

The oxygen consumption rates of the two controls (run at the start and at the end of the test) differed only by 1.5%.
Results with reference substance (positive control):
See table 2 in "Any other information on results incl. tables"
- Results with reference substance valid? yes
- Relevant effect levels: The 3-hour EC50 of the reference item 3,5-dichlorophenol was calculated to be 17 mg/L (the 95% confidence limits could not be calculated). The 3-hour EC50 is within the guideline-recommended range of 5–30 mg/L, confirming suitability of the activated sludge used.
- Other: none
Reported statistics and error estimates:
None

Table 6.1.7/2: Influence of the test item and 3,5 -Dichlorophenol (reference item) on the oxygen consumption of activated sludge

Vessel no.

Nominal concentration of test chemical (mg/L)

Oxygen consumption rate (mg O2/L/min)

Inhibition (%)

pH values

Oxygen concentration (mg O2/L)

Start*

End*

Start*

End*

control

A

B

0

0

1.305

1.286

 

7.4

7.6

8.2

8.2

8.0

7.9

8.4

8.4

Mean

Deviation (%)

1.295

1.5

 

Test item

4

100

1.277

1.4

7.7

8.0

8.4

8.3

Reference item

1

2

3

5

16

50

1.110

0.716

0.138

14.3

44.7

89.3

7.5

7.7

7.7

8.2

8.2

8.2

8.1

8.2

8.3

8.4

8.4

8.4

* Start and end of the 3 -hour incubation period

Validity criteria fulfilled:
yes
Conclusions:
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 100 mg/L. The 3-hour NOEC was at least 100 mg/L and the 3-hour EC50 could not be calculated but was clearly higher than 100 mg/L (nominal concentration). In conclusion, no effect up to water solubility limit of the test item.
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 Regulation 440/2008, Part C.11, and the OECD Guideline for Testing of Chemicals, No. 209.

In accordance with the EU Commission Regulation 440/2008 (C.1-C.3), a limit test with a nominal test concentration of 100 mg/L was performed. 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 100 mg/L. The saturation concentration (water solubility limit of the test item under the test conditions) was reached and no inhibitory effect was determined after three hours contact time. Thus, the 3-hour NOEC of the test item to activated sludge microorganisms was at least 100 mg/L. This value might even be higher but concentrations above 100 mg/L were not tested. The 3-hour EC50 could not be calculated but was clearly higher than 100 mg/L.

Description of key information

EU Method C.11, OECD Guideline 209, GLP, key study, validity 1:

No inhibitory effect was determined after three hours contact time at 100 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
100 mg/L

Additional information

One key study was available, according to the EU Commission Regulation 440/2008, Part C.11, and the OECD Guideline for Testing of Chemicals, No. 209 with GLP statement, on a 3-hour respiration inhibition test to assess the inhibitory effect of the substance on the respiration rate of aerobic wastewater microorganisms of activated sludge.

In accordance with the EU Commission Regulation 440/2008 (C.1-C.3), a limit test with a nominal test concentration of 100 mg/L was performed. 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 100 mg/L. The saturation concentration (water solubility limit of the test item under the test conditions) was reached and no inhibitory effect was determined after three hours contact time. Thus, the 3-hour NOEC of the test item to activated sludge microorganisms was at least 100 mg/L. This value might even be higher but concentrations above 100 mg/L were not tested. The 3-hour EC50 could not be calculated but was clearly higher than 100 mg/L.