Registration Dossier

Ecotoxicological information

Toxicity to microorganisms

Currently viewing:

Administrative data

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

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guidelineopen allclose all
according to
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
according to
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
GLP compliance:
yes (incl. certificate)

Test material


Test solutions

Details on test solutions:
The Result of a preliminary solubility trial showed that VRT-126017 dcha was insufficiently soluble to allow the preparation of a suitable aqueous
stock solution for either test. Therefore, at test initiation appropriate weights were established in one-litre test beakers, dechlorinated tap water
(284 ml) was added and the mixtures then stirred on a magnetic stirrer for one hour and twenty five minutes in order to form dispersions.

Test organisms

Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
A sample of activated sludge was obtained the day before the start of the tests from the aeration basin at Worlingworth Sewage Treatment Works,
which treats predominantly domestic waste. In the laboratory, the samples were maintained under aerobic conditions until required. The
concentration of suspended solids in a homogenised sample was determined on the day of collection and immediately before the start of the tests.
On the day of collection, aliquots (25 ml) of the activated sludge were filtered through dried and preweighed Whatman GF/C filter papers which were
then dried again at approximately 105°C for at least one hour, allowed to cool in a desiccator and reweighed. The mixed liquor suspended solids
(MLSS) content of the activated sludge was then calculated. In the definitive test the MLSS was adjusted to 4 g/l by the removal of supernatant.
Synthetic sewage (50 ml/l) was added to each stock of activated sludge and these were aerated overnight. On the day of the test, the MLSS content of
the sludge was determined and adjusted to 4 g/l by the addition of dechlorinated tap water. The pH and temperature of the sludge was also measured.

Study design

Water media type:
Limit test:
Total exposure duration:
3 h

Test conditions

200-250 mg/L as CaCO3
Nominal and measured concentrations:
preliminary range finding test at nominal concentrations of 10, 100 and 1000 mg/l
second (definitive) test at nominal concentrations of 500, 1000, 2000, 4000 and 8000 mg/l
Details on test conditions:
The prepared mixtures were aerated for three hours in a thermostatically-controlled water bath, using a glass aerator connected to a laboratory
supply of oil-free compressed air (one litre/minute).Following the exposure period, a well-mixed sample of each mixture was transferred to a
biochemical oxygen demand (BOD) bottle (nominal capacity, 270 ml). The rate of oxygen consumption was measured, over a period of approximately ten minutes or until the dissolved oxygen concentration fell below 2 mgO2/l using a Yellow Springs Instrument (YSI) dissolved oxygen meter, with
temperature probe and self stirring bottle probe, connected to a chart recorder. The pH and temperature of the samples were measured at the start
and end of the test.
Reference substance (positive control):

Results and discussion

Effect concentrations
3 h
Dose descriptor:
Effect conc.:
4 320 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
respiration rate
Details on results:
The EC50 of the test substance was calculated from the results of the definitive test to be 4320 mg/l (95% confidence limits, 2760 - 7900 mg/l). The
EC20 value was calculated to be 703 mg/l (95% confidence limits, 231 - 1500 mg/l).
Results with reference substance (positive control):
Sludge respiration rates were progressively reduced in the presence of increasing concentrations of 3,5-DCP. The three-hour 50% effect
concentrations (EC50) for 3,5-DCP was calculated to be 16.3 mg/l (95% confidence limits, 9.5 - 34.7 mg/l) in the preliminary test, and 12.0 mg/l (95% confidence limits, 7.4 - 20.0 mg/l) in the definitive test.

Applicant's summary and conclusion

The EC50 of the test substance was calculated to be 4320 mg/l (95% confidence limits, 2760 - 7900 mg/l). The EC20 value was calculated to be 703 mg/l (95% confidence limits, 231 - 1500 mg/l).