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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 Oct 2000 to 25 Oct 2000
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
Version / remarks:
April 1984
Qualifier:
according to guideline
Guideline:
other: EEC L133 s. 118-122
Version / remarks:
30 May 1988
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
200.3 mg test item was mixed (intensivly stirred for 15 min) and made up to 1000 mL with water. The stock solution (suspension) was freshly prepared at the start of the test. 200.6 mg reference item were mixed with 4.0 mL NaOH (1 N) and 12 mL water (dest). The pH of this stock solution was adjusted with H2SO4 to 7.8 and made up to 1000 mL water.
Test organisms (species):
activated sludge
Details on inoculum:
- Preparation of inoculum for exposure: The sludge was separated from the aqueous layer by settling instead of The pH of the sludge before use was 7.1.
- Initial biomass concentration: The concentration of the sludge in the test bottles was 1.72 g suspended solids/L.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
20 ± 1°C
pH:
7.9 after collection; 7.1 after preparation (before use)
The pH of the mixtures in the test bottles was 8.4 - 8.5 at the end of exposure time.
Nominal and measured concentrations:
Nominal concentrations: 1.0, 3.2, 10.0, 32.0, and 100 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 ml BOD flasks with gas inlet
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 2
- Nutrients provided for bacteria: 16 g Peptone, 11 g Meat extract, 3.0 g Urea, 0.7 g NaCl, 0.4 g CaC12 - 2 H20, 0.2 g MgSO4 7 H20, 2.8 g K2HP04 were dissolved and the volume made up to liter with dechlorinated drinking water.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dechlorinated drinking water

EFFECT PARAMETERS MEASURED:
Oxygen consumption per hour in mg/L with a microprocessor ionalizer and plotted on a recorder.

TEST CONCENTRATIONS
- Test concentrations: 100, 32.0. 10.0, 3.2 and 1.0 mg/L
Reference substance (positive control):
yes
Remarks:
3.5-Dichlorophenol; 32.0, 10.0 and 3.2 mgIL
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
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 on result:
other: No significant inhibition of total respiration was observed at any test concentration.
Details on results:
- Any observations that might cause a difference between measured and nominal values: The test item was not homogeneously distributed in the test vessels. At the end of exposure small drops were sticking on the surface of the test vessels.
- Blank controls oxygen uptake rate: 44.2 - 46.4 mg/L
- Variation of oxygen uptake rate in control replicates: 4%
Results with reference substance (positive control):
For the reference item (tested nominal concentrations: 3.2, 10.0 and 32.0 mg/L):
EC50(3h) : 9.1 mg/L
EC20(3h) : 3.0 mg/L
EC80(3h) 27.8 mg/L:
Reported statistics and error estimates:
The inhibitory values were calculated on the basis of the measured time dependend oxygen consumption of a blank and test solution.
The Results ( ECxx Values ) were determined after calculation of the linear regression.

Table 1.Oxygen consumption rate and inhibitory effects of the test item and reference

Sample

Concentration (mg/L)

Consumption rate (mg/L/h)

Inhibition (%)

pH

Blank 1

0.0

46.4

-2

8.4

Blank 2

0.0

44.2

2

8.5

Reference 1

32.0

8.3

82

8.4

Reference 2

10.0

19.6

57

8.5

Reference 3

3.2

36.5

20

8.5

Test conc. 1

100.0

34.4

24

8.5

Test conc. 2

32.0

40.1

11

8.5

Test conc. 3

10.0

44.5

2

8.4

Test conc. 4

3.2

42.8

5

8.5

Test conc. 5

1.0

44.9

1

8.5

The pH was measured after 3h aeration.

Negative inhibition results, when obtained in a test, are a consequence of the comparison of the oxygen consumption values obtained in the blank, and the higher oxygen consumption values obtained in the presence of the test item, which are due to biological variations between the various test samples.

Validity criteria fulfilled:
yes
Conclusions:
In an activated sludge respiration inhibition test, performed according to OECD TG 209, the EC 50 (3h) for the test substance was determined to be > 100 mg/L. Based on the inherent toxicity of the test substance in this test alone, the test item can be classified as not toxic to bacteria.
Executive summary:

The purpose of this test performed following OECD TG 209 and in compliance with GLP was to determine the effect of the test substance on aerobic waste-water bacteria. The respiration rate of an activated sludge fed with a standard amount of synthetic sewage feed was measured after a contact time of 3 hours. The respiration rate of the same activated sludge was also measured in the presence of 1.0. 3.2, 10.0, 32.0 and 100.0 mg test item/L and 3.2, 10.0 and 320 mg/L reference item, respectively. The sludge concentration in the test bottles was 1.72 g suspended solids/L. The test with the test substance was conducted at a temperature of 20±1°C. The pH of sludge was 7.9 after collection and 7.1 after preparation (before use). The pH of the mixtures in the test bottles was 8.4 - 8.5 at the end of exposure time. The EC50 (3h) of the reference item 3,5-Dichlorphenol was 9.1 mg/L. The inhibitory effect of the test item was expressed as a percentage of the mean respiration rates of two controls. The EC 50 (3h) for the test substance is >100 mg/L. No significant inhibition of total respiration was observed at any test concentration, therefore the NOEC for the test substance is assumed to be > 100 mg/L. Based on the inherent toxicity of the test substance in this test alone, the test item can be classified as not toxic to bacteria.

Description of key information

3-h EC50 > 100 mg/L, Activated sludge, inhibition of total respiration, OECD TG 209, Grade 2001

3-h NOEC > 100 mg/L, Activated sludge, inhibition of total respiration, OECD TG 209, Grade 2001

Key value for chemical safety assessment

EC50 for microorganisms:
100 mg/L

Additional information

OECD TG 209, Grade 2001

The purpose of this test performed following OECD TG 209 and in compliance with GLP was to determine the effect of the test substance on aerobic waste-water bacteria. The respiration rate of an activated sludge fed with a standard amount of synthetic sewage feed was measured after a contact time of 3 hours. The respiration rate of the same activated sludge was also measured in the presence of 1.0. 3.2, 10.0, 32.0 and 100.0 mg test item/L and 3.2, 10.0 and 320 mg/L reference item, respectively. The sludge concentration in the test bottles was 1.72 g suspended solids/L. The test with the test substance was conducted at a temperature of 20±1°C. The pH of sludge was 7.9 after collection and 7.1 after preparation (before use). The pH of the mixtures in the test bottles was 8.4 - 8.5 at the end of exposure time. The EC50 (3h) of the reference item 3,5-Dichlorphenol was 9.1 mg/L. The inhibitory effect of the test item was expressed as a percentage of the mean respiration rates of two controls. The EC 50 (3h) for the test substance is >100 mg/L.No significant inhibition of total respiration was observed at any test concentration, therefore the NOEC for the test substance is assumed to be > 100 mg/L.