Registration Dossier

Administrative data

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07-nov-2008 to 21-nov-2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
ISO 8192 (Water quality - Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): ASA404 C7
- Substance type: Off white powder
- Physical state: Solid
- Analytical purity: 99.9%
- Lot/batch No.: 072301
- Expiration date of the lot/batch: 18 March 2009
- Stability under test conditions: Stable
- Storage condition of test material: At room temperature in the dark

Sampling and analysis

Analytical monitoring:
no

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
Method- Since ASA404 C7 was hardly soluble in water test concentrations were prepared separately in Milli-RO water (tap water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA). One litre test bottles were filled with 250 ml of test substance mixtures with initial loading rates twice as high as the final test concentration. These mixtures were stirred in closed bottles for 25-27 hours. Subsequently, 16 ml synthetic sewage feed, 200 ml sludge and Milli-RO water up to 500 ml were added resulting in the final loading rates (see also section 6.4). Optimal contact between the test substance and test medium was ensured applying continuous aeration and stirring.

- Controls: blank and reference substance

Test organisms

Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
The sludge was coarsely 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 and 4.4 g/l of sludge, as used for experiment 1 and 2, respectively). The pH was 7.8 on the day of testing in both experiments. 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.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Remarks on exposure duration:
Oxygen consumption was measured and recorded for approximately 10 minutes after the 3 hours treatment period.

Test conditions

Test temperature:
first study: 19.1 - 19.6°C
second study: 19.0 - 20.6°C
pH:
first study: 6.3 - 8.1
second study: 6.1 - 8.0
Dissolved oxygen:
Oxygen consumption
first study: 14 - 17 mg O2/l/h
second study: 11 - 54 mg O2/l/h
Nominal and measured concentrations:
First study: nominal concentration: 1000 mg/l
Second study: nominal concentrations: 100, 180, 320, 560 and 1000 mg/l
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type: open
- Material, size, headspace, fill volume: All glass, approximately 300 ml oxygen bottles (measuring) and 1 litre test bottles (exposure)
- Aeration: during exposure with clean, oil-free air
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 2
- No. of vessels per vehicle control (replicates): not applicable
- Biomass loading rate: 1.8 g/l suspended solids


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap-water purified by reverse osmosis (Milli-RO)

EFFECT PARAMETERS MEASURED :
-The respiration rate from each vessel, in mg O2/l/hr, was calculated from the linear part of the respiration curve, which was generally between 2.5 and 6.5 mg O2/l.

TEST CONCENTRATIONS
- First study: Test concentrations: 1000 mg/l. Results used to determine the conditions for the definitive study: yes
- Second study: Spacing factor for test concentrations: geometric progression with a factor 1.8
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol

Results and discussion

Effect concentrations
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
>= 320 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
In the first experiment 62 and 69% inhibition of the respiration rate of the sludge was recorded at a loading rate of 1000 mg ASA404 C7 per litre.

In the second experiment no significant inhibition of the respiration rate of the sludge was recorded up to a loading rate of 180 mg/l (based on nominal concentrations). At a loading rate of 320 and 560 mg/l very slight inhibition (13%) of respiration rate of the sludge was recorded. Whereas, 80% inhibition of the respiration rate of the sludge was recorded at a loading rate of 1000 mg/l.

The EC50 was estimated to be at a loading rate of 770 mg/l (based on nominal concentrations).
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: The EC50 of 3,5-dichlorophenol was 7.6 mg/l
Reported statistics and error estimates:
The EC50, EC20 and EC80 for the test/reference substance, was determined using linear regression analysis.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
It is concluded that this test is valid and that significant inhibition of respiration rate of the sludge was recorded at and above a loading rate of 320 mg/l (based on nominal concentrations).

The EC50 was estimated to be at a loading rate of 770 mg/l (based on nominal concentrations).