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Ecotoxicological information

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:
Jan - Apr 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
After the 3-hour contact time, the oxygen consumption was recorded for a period of 10 to 11 minutes.
The pH was determined in the remaining part of the reaction mixture. This procedure was repeated for all test/reference substance concentrations and controls.
The medium temperature was recorded continuously in temperature control vessels.
Vehicle:
yes
Remarks:
Milli-RO water (tap water purified by reverse osmosis)
Details on test solutions:
The test substance was not completely soluble in test medium at the loading rates initially prepared. Therefore, 1-Litre test bottles were filled with 200 ml of test substance mixtures in Milli-RO water (tap water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA) with initial loading rates of 2.5 times the final loading rate. These mixtures were stirred in closed dark brown bottles for
24 to 25¼ hours. Subsequently, 16 ml synthetic medium, 250 ml sludge and Milli-RO water up to 500 ml were added resulting in the required loading rates. Optimal contact between the test substance and test organisms was ensured applying continuous aeration and stirring.

Test system: Micro-organisms in activated sludge.

Adjusted ISO medium, formulated using RO-water (tap-water purified by reverse osmosis; GEON Waterbehandeling, Berkel-Enschot, The Netherlands) with the following composition:
CaCl2.2H2O 211.5 mg/l
MgSO4.7H2O 88.8 mg/l
NaHCO3 46.7 mg/l
KCl 4.2 mg/l
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
Test system
Micro-organisms in activated sludge.
Source
Municipal sewage treatment plant: 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.
Number of micro-organisms Number of micro-organisms was determined as the amount of Mixed Liquor Suspended Solids (MLSS) per litre test medium.
Preparation of the sludge
The sludge was coarsely sieved (1 mm), washed and diluted with ISO-medium. A small amount of the sludge was weighed and dried overnight at ca. 105°C to determine the amount of suspended solids (3.0 g/l of sludge, as used for the test). The pH was 7.7 on the day of testing. The batch of sludge was used one day after collection; therefore 50 ml of synthetic medium was added per litre of activated sludge at the end of the collection day. The sludge was kept aerated at test temperature until use.
Medium
Adjusted ISO medium, formulated using RO-water (tap-water purified by reverse osmosis; GEON Waterbehandeling, Berkel-Enschot, The Netherlands) with the following composition:
CaCl2.2H2O 211.5 mg/l
MgSO4.7H2O 88.8 mg/l
NaHCO3 46.7 mg/l
KCl 4.2 mg/l
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Post exposure observation period:
oxygen consumption was recorded for 10 to 11 minutes
Test temperature:
19.4 and 22.7°C
pH:
The pH in the controls, before addition of the sludge, was between 7.2 and 7.6
After the 3 hour exposure period the pH was between 7.8 and 8.2.
The pH in all test vessels containing test substance, before addition of sludge was between 5.4 and 7.5. After the 3 hour exposure period the pH was between 7.0 and 7.9.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
1 000 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Results with reference substance (positive control):
The batch of activated sludge was checked for sensitivity by testing the reference substance 3,5-dichlorophenol, which showed normal sensitivity.
Validity criteria fulfilled:
yes
Conclusions:
under the conditions of this present test substance was not toxic to waste water (activated sludge) bacteria at or below a loading rate of 1000 mg/l (NOEC).

Description of key information

Under the conditions of this present test substance was not toxic to waste water (activated sludge) bacteria at or below a loading rate of 1000 mg/l (NOEC).

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L
EC10 or NOEC for microorganisms:
1 000 mg/L

Additional information