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Toxicity to microorganisms

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Reference
Endpoint:
toxicity to microorganisms
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 May 1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Version / remarks:
1988
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Version / remarks:
1984
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
Stability under storage conditions: stable
Stability in water: not indicated
Solubility in water: <2.4 mg/L at 20 °C
Analytical monitoring:
no
Vehicle:
yes
Remarks:
Tween-80
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION:
- Method: a suspension of 0.5 g/L was prepared using Tween-80 as additive. Ultra sonication was used to accelerate dissolving and to ensure homogeneity. 100 mg/L, corresponding to 100 mL of the prepared stock solution in 500 mL final volume, was tested in duplicate.
- Controls: for each of the two tests: 2 controls without test substance or additives and 2 controls without test substance but containing Tween-80.
- Chemical name of vehicle: Tween-80
- Concentration of vehicle in test medium: 0.006% (v/v)
Test organisms (species):
activated sludge
Details on inoculum:
- Name and location of sewage treatment plant where inoculum was collected: Municipal sewage treatment plant: 'Waterschap de Dommel' , 's-Hertogenbosch, the Netherlands
- Preparation of inoculum for exposure: the sludge was coarsly sieved and washed with tap-water. A small amount of the sludge was weighed and dried at ca. 105°C. From this result the amount of suspended solids was calculated: the level was 3.5 g/L of sludge. The batch of sludge was used on subsequent days (maximum four days), therefore 50 mL of synthetic sewage feed was added to each litre of activated sludge at the end of each working day. The sludge was kept aerated at 20 +/- 2°C until use. Before use, the pH was checked (7.6).
- Initial biomass concentration: the amount of Mixed Liquor Suspended Solids (MLSS) per litre test medium was 3.5 g/L of sludge.
Test type:
other: aerobic
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
0.5 h
Test temperature:
20 +/- 2°C
pH:
Ranging from 7.9 to 8.1 between flasks.
Details on test conditions:
TEST SYSTEM
- Test vessel: all-glass, 500 mL beakers and 300 mL oxygen bottles
- Air supply: clean, oil-free air
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
- No. of vessels per vehicle control (replicates): 2
- Sludge concentration: 1.4 g/L
- Nutrients provided for bacteria: synthetic sewage feed

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Milli-Q water

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod/light intensity: not indicated
- Details on termination of incubation: not indicated

EFFECT PARAMETERS MEASURED: oxygen consumption was measured for 10 minutes after 30 minutes of exposure time

TEST CONCENTRATIONS
- Spacing factor for test concentrations: n.a., limit test of 100 mg/L
- Reference study concentrations: 3.2, 10, 32 mg/L
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Key result
Duration:
0.5 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:
0.5 h
Dose descriptor:
EC10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Details on results:
- No abnormalities observed
- Blank controls oxygen uptake rate: 42 mg O2/L/hr and 40 mg O2/L/hr
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- Relevant effect levels: EC50 = 14.55 mg/L, 95%-CI (11.68, 18.73 mg/L)

For more detailed results, refer to attached document (CRL 137824 - Tables)

Validity criteria fulfilled:
yes
Remarks:
1) Respiration rates of both controls were within 15% of each other (i.e. 10% and 9% for additive controls) . 2) The EC50 of 3,5-dichlorophenol was in the accepted range of 5-30 mg/L (i.e. 15 mg/L).
Conclusions:
Under the conditions of this present test Alcamizer 5 was not toxic to waste-water bacteria.
Executive summary:

This study was performed to assess the effect of Alcamizer 5 on the respiration rate of activated sludge. The study was conducted in accordance with OECD 209 and under GLP conditions.

A solution of 100 mg/L, taken from a stock solution of 0.5 g/L, was tested in duplicate. Tween-80 was used as an additive because the test substance was hardly soluble in water.

No significant inhibition in respiration rate of sludge (>10%) was recorded at a test concentration of 100 mg/L. The duplicate measurement confirmed the result of the first measurement. Therefore, no further testing was needed.

The validity criteria were met and the reference test was considered to be valid.

Under the conditions of this present test Alcamizer 5 was not toxic to waste-water bacteria and thus the EC10 and EC50 values are > 100 mg/L

Description of key information

A study was performed to assess the effect of Alcamizer 5 on the respiration rate of activated sludge. The study was conducted in accordance with OECD 209 and under GLP conditions.

A solution of 100 mg/L, taken from a stock solution of 0.5 g/L, was tested in duplicate. Tween-80 was used as an additive because the test substance was hardly soluble in water.

No significant inhibition in respiration rate of sludge (>10%) was recorded at a test concentration of 100 mg/L. The duplicate measurement confirmed the result of the first measurement. Therefore, no further testing was needed.

The validity criteria were met and the reference test was considered to be valid.

Under the conditions of this present test Alcamizer 5 was not toxic to waste-water bacteria and thus the EC10 and EC50 values are > 100 mg/L.

Key value for chemical safety assessment

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