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Toxicity to microorganisms

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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2004-09-22
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Version / remarks:
1984
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Details on sampling:
No concentrations of the test substance were measured.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: 1000 mg test item were filled up to 200 mL with deionised water and completely dissolved by stirring. A stock-solution concentration of 5 g/L was obtained. One test solution with a final volume of 500 mL was tested per treatment in a glass flask (>500 mL). Samples were prepared in about 15-minute intervals.
- Controls: blank control (without test substance) and positive control performed with the reference substance 3,5-dichlorophenol .
- Evidence of undissolved material: no
Test organisms (species):
activated sludge, domestic
Details on inoculum:
- Name and location of sewage treatment plant where inoculum was collected: Sewage plant Gross-Zimmern, Germany
- Preparation of inoculum for exposure: The sludge was washed by centrifugation and the supernatant liquid phase was decanted. The solid material was resuspended in tap water and again centrifuged. The latter procedure was repeated twice. An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge (g) to its dry weight (g) determined. Based on this ratio, calculated aliquots of washed sludge suspension, corresponding to 3 g dry material per litre, were made up with tap water.
- pH: The pH of the activated sludge was 7.5.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
180 min
Post exposure observation period:
Not performed
Hardness:
No data
Test temperature:
21 - 22 °C
pH:
7.5
Dissolved oxygen:
6.5 - 2.5 mg O2/L
Salinity:
Not applicable
Conductivity:
Not applicable
Nominal and measured concentrations:
- Nominal concentration: 10, 32, 100, 320 and 1000 mg/L
- Measured concentrations: No concentrations were measured.
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass flasks of approximately 1 litre volume and Karlsruher flasks of 250 mL volume.
- Fill volume: 500 mL
- Aeration: yes
- No. of vessels per concentration (replicates): 5
- No. of vessels per blank control (replicates): 1
- No. of vessels per positive control (replicates): 3
- No. of vessels per abiotic control (replicates): 1
- Sludge concentration (weight of dry solids per volume): 3 g/L


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to guideline OECD 209, 1984

OTHER TEST CONDITIONS
- Adjustment of pH: No

EFFECT PARAMETERS MEASURED: Inhibition of respiration rate of activated sludge after 3 hours.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.125
- Range finding study : not performed
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Key result
Duration:
180 min
Dose descriptor:
NOEC
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of heterotrophic respiration
Key result
Duration:
180 min
Dose descriptor:
EC50
Effect conc.:
17.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of heterotrophic respiration
Remarks on result:
other: result calculated with a 95 % confidence limit of 12.4 to 22.5 mg/L
Duration:
180 min
Dose descriptor:
other: EC20
Effect conc.:
4.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of heterotrophic respiration
Remarks:
95 % C.L. (1.9 - 6.6 mg/L)
Remarks on result:
other: result calculated with a 95 % confidence limit of 1.9 to 6.6 mg/L (1.9 - 6.6 mg/L)
Details on results:
- Any observations that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no
- Adsorption: no
- Inhibition of respiration rate: In comparison to the inoculum controls the respiration rate of the activated sludge was inhibited by 38.2 and 61.8 % at the two lowest nominal concentrations of 10 and 32 mg/L, respectively. At the next higher nominal concentrations of 100 and 320 mg/L, the respiration rate was inhibited by 88.2 and 94.1 %, respectively, and reached a plateau at the highest nominal test concentration of 1000 mg/L with an inhibition rate of 97.1 %.
- 3-hour EC20 and 3-hour EC50: The 3-hour EC20 was calculated by linear regression analysis using the inhibition rates of the 1000, 320, 100, 32 and 10 mg/L test concentrations, and was calculated to be 4.2 mg/L nominal with a 95 % confidence limit of 1.9 to 6.6 mg/L. The 3-hour EC50 was calculated by linear regression analysis using the inhibition rates of the 1000, 320, 100, 32 and 10 mg/L test concentrations, and was calculated to be 17.3 mg/L nominal with a 95 % confidence limit of 12.4 to 22.5 mg/L.
Results with reference substance (positive control):
The 3-hour EC 50 of 3,5-Dichlorophenol was calculated to be 6.1 mg/L.
Validity criteria fulfilled:
yes
Conclusions:
The toxic effects of the test substance on the oxygen consumption rate of aerobic micro-organisms (activated sludge) after of 180 min was determined according to the OECD 209 (1984). The EC50 was determined to be 17.3 mg/L and the NOEC was determined to be 10 mg/L.
Executive summary:

The objective of this key study according to OECD 209 (1984) under GLP conditions was to determine the toxic effects of the test substance on the oxygen consumption rate of aerobic micro-organisms (activated sludge) after short-term exposure of 180 min, compared with the blank control. The test was performed in nominal concentrations of 0 (control), 10, 32, 100, 320 and 1000 mg/L. No concentrations were measured. The inoculum was activated sludge from a municipal waste treatment plant of Gross-Zimmern in Germany. The sludge concentration was 3 g/L (dry weight) and a mineral medium according to the guideline was used. A reference substance (3,5 dichlorphenol) was used as positive control. The EC50 value of the reference substance 3,5-dichlorphenol was determined to be 6.1 mg/L, which was within the acceptable limits, (5.0 to 30 mg/L) as specified in OECD 209 Guideline. The respiration rates of the two controls did not differ by more than 15 % (measured value: 5.7 %). The 3-hour EC 50 of the reference item 3,5-Dichlorophenol was determined to be 6.1 mg/L and is thus in the range of 5 to 30 mg/L for the used activated sludge batch. The concentration of dissolved oxygen did not drop below 2.5 mg O2/L during the incubation period, and just before the measurements of the respiration rates the oxygen concentrations were at least 7.3 mg O2/L and thus > 6.5 mg O2/L. All validation criteria were fulfilled. The EC50 for the test substance was determined to be 17.3 mg/L and the NOEC for the test substance was determined to be 10 mg/L. 

Description of key information

The toxic effects of the test substance on the oxygen consumption rate of aerobic micro-organisms (activated sludge) after of 180 min was determined according to the OECD 209 (1984), (reference 6.1.7-1). The EC50 was determined to be 17.3 mg/L and the NOEC was determined to be 10 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:
17.3 mg/L
EC10 or NOEC for microorganisms:
10 mg/L

Additional information

The objective of this key study according to OECD 209 (1984) under GLP conditions was to determine the toxic effects of the test substance on the oxygen consumption rate of aerobic micro-organisms (activated sludge) after short-term exposure of 180 min, compared with the blank control. The test was performed in nominal concentrations of 0 (control), 10, 32, 100, 320 and 1000 mg/L. No concentrations were measured. The inoculum was activated sludge from a municipal waste treatment plant of Gross-Zimmern in Germany. The sludge concentration was 3 g/L (dry weight) and a mineral medium according to the guideline was used. A reference substance (3,5 dichlorphenol) was used as positive control. The EC50 value of the reference substance 3,5-dichlorphenol was determined to be 6.1 mg/L, which was within the acceptable limits, (5.0 to 30 mg/L) as specified in OECD 209 Guideline. The respiration rates of the two controls did not differ by more than 15 % (measured value: 5.7 %). The 3-hour EC 50 of the reference item 3,5 -dichlorophenol was determined to be 6.1 mg/L and is thus in the range of 5 to 30 mg/L for the used activated sludge batch. The concentration of dissolved oxygen did not drop below 2.5 mg O2/L during the incubation period, and just before the measurements of the respiration rates the oxygen concentrations were at least 7.3 mg O2/L and thus > 6.5 mg O2/L. All validation criteria were fulfilled. The EC50 for the test substance was determined to be 17.3 mg/L and the NOEC for the test substance was determined to be 10 mg/L.