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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31 Mar - 21 May 2003
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Deviations from the current guideline. For details refer to 'Test guideline'.
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Version / remarks:
1984
Deviations:
yes
Remarks:
Tap water is not specified. 2 replicates for controls and 1 replicate for each substance conc. were tested (OECD 209: 6 control replicates and 5 treatment conc. replicates. Oxygen concentrations were above the recommendation of 2.0 - 7.0 mg/L.
Qualifier:
according to guideline
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Version / remarks:
1984
GLP compliance:
yes (incl. QA statement)
Remarks:
Swiss Federal Office of Public Health, Switzerland
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: According to the results of a pre-test, no stock solution could be prepared due to the low water solubility of the test item. Therefore, test item amounts of 4.98, 16.2, 49.9, 160.5 and 500.2 mg were directly weighed (by means of an analytical balance) into the test flasks. Then 284 mL tap water was added to each flask. The test item was mixed into the tap water by 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. Then, 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:
- Name and location of sewage treatment plant where inoculum was collected: ARA Ergolz II, Fiiliinsdorf, Switzerland
- Method of cultivation: During the holding of two days prior to use, the sludge was fed daily with 50 mL synthetic sewage feed according to OECD 209 per liter and was kept at room temperature under continuous aeration until use.
- Preparation of inoculum for exposure: 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
- Initial biomass concentration: 3.7 g/L dry weight
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
20 °C
pH:
7.0 - 8.2
Dissolved oxygen:
8.2 - 9.0 mg/L
Nominal and measured concentrations:
Nominal concentrations: 0 (control), 10, 32, 100, 320 and 1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 1000 mL flasks
- Type (delete if not applicable): open / closed
- Material, headspace, fill volume: glass, 500 mL, 500 mL
- Aeration: yes
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 2
- Sludge concentration (weight of dry solids per volume): 1.48 g dry material/L test medium
- Nutrients provided for bacteria: 16 mL synthetic sewage feed according to OECD 209
- Biomass loading rate: 400 mL activated sludge/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: tap water
- Particulate matter: not reported
OTHER TEST CONDITIONS
- Adjustment of pH: no
- Details on termination of incubation: After 3 hours of exposure, the well-mixed samples of each test medium was poured into a BOD-flask after exactly three hours incubation time, and was not further aerated.
EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Dissolved oxygen concentration was continuously recorded for about ten to fifteen minutes. During measurement, the samples were continuously stirred on a magnetic stirrer.
TEST CONCENTRATIONS
- Spacing factor for test concentrations: approximately 3.125 - 3.2
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
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
Details on results:
- Effect concentrations exceeding solubility of substance in test medium: yes, a part of the test item was suspended in the test media.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: EC50 (8 h): 8.5 mg/L which is within the acceptable limits, (5.0 to 30 mg/L) as specified in OECD 209 Guideline

Table 1: Influence of the test item on oxygen consumption of activated sludge

Flask no

Test chemical

Nominal test item concentration (mg/L)

Oxygen consumption rate

(mg O2/L min-1)

Inhibition (%)

pH values

Oxygen concentration

(mg O2/L)

1

Control

0

1.075

 

7.0

8.0

8.3

9.0

10

Control

0

1.027

 

7.1

8.2

8.3

8.5

5

Test item

10

1.085

-3.2

7.2

8.1

8.2

8.2

6

Test item

32

1.007

4.2

7.1

8.2

8.2

8.3

7

Test item

100

1.073

-2.1

7.0

8.0

8.4

8.2

8

Test item

320

1.080

-2.7

7.0

8.0

8.3

8.6

9

Test item

1000

1.087

-3.4

7.1

8.2

8.3

8.2

Table 2: Validity criteria for OECD 209

Criterion from the guideline

Outcome

Validity criterion fulfilled

The blank controls (without the test substance or reference substance) oxygen uptake rate should not be less than 20 mg oxygen per one gram of activated sludge (dry weight of suspended solids) in an hour.

 1.075 and 1.027 mg oxygen/L x min which is corresponding to

64.5 and 61.6 mg oxygen/1.48 g sludge x h which corresponds to

43.6 and 41.6 mg oxygen/g activated sludge in an hour

 yes

The coefficient of variation of oxygen uptake rate in control replicates should not be more than 30% at the end of definitive test.

 3.2%

 yes

Validity criteria fulfilled:
yes
Remarks:
For further details please refer to “Any other information on results incl. tables”.

Description of key information

EC50 (3 h) > 1000 mg/L (activated sludge, OECD 209, nominal)

NOEC (3 h) 1000 mg/L (activated sludge, OECD 209, nominal)

Key value for chemical safety assessment

Additional information

The effects 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 GLP and OECD 209.

Test item concentrations were tested at 10, 32, 100, 320 and 1000 mg/L. 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.

Down to the lowest test concentration of 10 mg/L a part of the test item was suspended in the test media. Thus, all test concentrations were clearly above the water solubility limit of the test item under the present test conditions. Up to and including the highest test concentration of 1000 mg/L the test item had no significant inhibitory effect (<15%) on the respiration rate of activated sludge after the incubation period of three hours. The inhibition of the respiration rates of the activated sludge after the incubation period were in the range of -3.4 to 4.2% compared to the controls. No concentration effect relationship was determined. Thus, the NOEC (3 h) was ≥ 1000 mg/L (nominal) and the derived EC50 (3 h) was determined to be > 1000 mg/L.