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activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Reference substance name:
.beta.-Cyclodextrin, 2-hydroxypropyl cycloheptaamylose
.beta.-Cyclodextrin, 2-hydroxypropyl cycloheptaamylose
Constituent 2
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Hill formula: (C42H70-nO35)(C3H7O)n; n(mittel)=5,25
2-Hydroxypropyl-.beta.-cyclodextrine ethers
Test material form:
solid: particulate/powder
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): hydroxypropylated .beta.-cyclodextrin
- Physical state: White powder
- Analytical purity: >95%
- Lot/batch No.: 74B008
- Expiration date of the lot/batch: 31 December 2002
- Stability in water: At least 96 h
- Stability under storage conditions: Stable
- Storage condition of test material: At room temperature in the dark

Sampling and analysis

Analytical monitoring:

Test organisms

Test organisms (species):
activated sludge
Details on inoculum:
- Laboratory culture: the accumulated biological mass produced in the treatment of waste water by the growth of bacteria and other micro-organisms in the presence of dissolved oxygen.
- Method of cultivation: no data
- Preparation of inoculum for exposure: sludge was coarsely sieved, washed and diluted with tap-water. After dilution a small amount of the
sludge was weighed and dried at ca. 105°C to determine the amount of suspended solids (3.7g/l of sludge, as used for the test). Before use the pH was checked.
- Pretreatment: 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 test temperature until use.
- Initial biomass concentration: no data

Study design

Test type:
Water media type:
Limit test:
Total exposure duration:
30 min

Test conditions

no data available
Test temperature:
Dissolved oxygen:
no data available
no data available
Nominal and measured concentrations:
3.2, 10, 32 and 100 mg/l (nominal)
Reference substance (positive control):
one at the start and one at the end

Results and discussion

Effect concentrations
30 min
Dose descriptor:
Effect conc.:
> 100 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
respiration rate
Details on results:
The EC50 of 3,5-dichlorophenol was 15 mg/I (regression line:Y = 71.01 X -33.25, Y =·% inhibition of respiration and X = log concentration (mg/I)).

Since all criteria for acceptability of the test were met, this study was considered to be valid.
Results with reference substance (positive control):
The mean respiration rate of control 1 and 2 was 38 mg O2/I/hr.The difference between the controls was 0%.
The mean respiration rate of control 2 and 3 was 41 mg O2/I/hr. The difference between the controls was 14.6%.

Applicant's summary and conclusion

Validity criteria fulfilled:
In conclusion, under the conditions of this present test, hydroxypropylated .beta.-cyclodextrin was not toxic to waste water (activated sludge) bacteria at a concentration of 100 mg/l.
Executive summary:

The influence of hydroxypropylated .beta.-cyclodextrinon the respiration rate of activated sludge was investigated after a contact time of 30 minutes.

The study procedure was based on OECD Guideline No. 209, adopted April 4, 1984 and EEC Directive 67/548 amended November 18, 1987 (87/302), Part C, Publication No. L133, adopted May 30, 1988.

Hydroxypropylated .beta.-cyclodextrinis white powder that was easily soluble in milli-Q water. A stock solution was prepared in milli-Q water at a concentration of 0.5 g/l. The pH of the clear stock solution was 7.6. A concentration of 100 mg/I, corresponding with 100 ml of the 0.5 g/l stock solution in 500 ml final volume, was tested in duplicate.

No significant inhibition in respiration rate of the sludge was recorded at 100 mg hydroxypropylated .beta.-cyclodextrinper litre. The duplicate measurement confirmed the result of the first measurement. Values of -7% and -5% inhibition were recorded (negative values indicate stimulation in respiration rate of the sludge). Hence, the EC50 for hydroxypropylated .beta.-cyclodextrinexceeded 100 mg/l and no further testing was necessary.

The respiration rates of the controls were within 15% of each other. The EC50 of the reference substance, 3,5 -dichlorophenol, was 15 mg/l. Therefore, the test was considered to be valid.

In conclusion, under the conditions of this present test,hydroxypropylated .beta.-cyclodextrinwas not toxic to waste water (activated sludge) bacteria at a concentration of 100 mg/l.