Registration Dossier

Administrative data

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study, no deviations

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent

Sampling and analysis

Analytical monitoring:
no

Test solutions

Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Since the substance was hardly soluble in water test concentrations were prepared separately in Milli-RO water. Two 1 litre test bottles were filled with 250 ml of test substance mixtures with initial loading rates of 200 mg/l. These mixtures were stirred in dark glass closed bottles for 24 hours.
Subsequently, 16 ml synthetic sewage feed, 200 ml activated sludge and Milli-RO water up to 500 ml were added resulting in a loading rate of 100 mg/l. This loading rate was tested in duplicate. Optimum contact between the test substance and test medium was ensured applying continuous aeration and stirring.

Activated sludge:
Source: Municipial sewage treatment plant (Waterschap de Maaskant, The Netherlands), receiving predominantly domestic sewage.
The sludge was sieved, 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 (3.8 g/l of sludge, as used for the test). The pH was 7.9 at the day of the testing. The batch of sludge was used one day after collection; therefore 50 ml of synthetic sewage feed was added per litre of activated sludge at the end of the collection day. The sludge was kept aerated at test temperature until use.

Composition of ISO Medium:
CaCl2.2H2O 211.5 mg/l
MgSO4.7H2O 88.8 mg/l
NaHCO3 46.7 mg/l
KCl 4.2 mg/l
Dissolved in tap water, purified by reverse osmosis

Composition of synthetic sewage feed:
16 g peptone
11 g meat extract
3 g urea
0.7 g NaCl
0.4 g CaCl.2H2O
0.2 g MgSO4.7H2o
2.8 g K2HPO4
Dissolved in Milli-Q water, made up to one litre and filtered

- Eluate:
- Differential loading:
- Controls:
- Chemical name of vehicle (organic solvent, emulsifier or dispersant):
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)):
- Evidence of undissolved material (e.g. precipitate, surface film, etc):

Test organisms

Test organisms (species):
activated sludge of a predominantly domestic sewage

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h

Test conditions

Test temperature:
20.1 - 20.5 °C
pH:
8.1
Nominal and measured concentrations:
Nominal concentrations were used
Details on test conditions:
TEST SYSTEM
Test vessel: Dark glass bottles, 1 litre
- Aeration: continuously during 3 h contact time

- Renewal rate of test solution: no
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 1

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Milli-RO water (tap water purified by reverse osmosis)
OTHER TEST CONDITIONS
- Adjustment of pH: no

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : After the 3 h contact time , a well mixed sample of the contents was poured into 300 ml oxygen bottle, and the flask was sealed with an oxygen electrode connected to a recorder, forcing the air out of the vessel.. Oxygen consumption was measured and recorded for approximately 10 minutes.
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol

Results and discussion

Effect concentrations
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The substance was not toxic to waste water (activated sludge) bacteria at a loading rate of 100 mg/l (based on nominal concentrations).
Executive summary:

The toxicity of the substance towards waste water bacteria was assessed in an assay according to OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test, 1984). Since the substance was hardly soluble in water test concentrations were prepared separately in Milli-RO water. Two 1 litre test bottles were filled with 250 ml of test substance mixtures with initial loading rates of 200 mg/L. These mixtures were stirred in dark glass closed bottles for 24 hours. Subsequently, 16 ml synthetic sewage feed, 200 ml activated sludge and Milli-RO water up to 500 ml were added resulting in a loading rate of 100 mg/L. This loading rate was tested in duplicate. Optimal contact between the test substance and test medium was ensured applying continuous aeration and stirring. Thereafter oxygen consumption was measured and recorded for approximately 10 minutes.

No significant inhibition of respiration rate of the sludge was recorded at a loading rate of 100 mg/L. The duplicate measurement confirmed the results of the first measurement. Therefore no further testing was necessary.

The EC50 of the substance exceeded a loading rate of 100 mg/L (based on nominal concentrations).