Registration Dossier

Administrative data

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04-mar-2010 to 19-mar-2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This 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:
2010
Report Date:
2010

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): Blown rapeseed oil
- Substance type: Yellow to brown viscous liquid
- Physical state: liquid
- Analytical purity: treated as 100% pure
- Expiration date of the lot/batch: 16 December 2010
- 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:
The batch of Blown rapeseed oil tested was a yellow to brown viscous liquid and 100% pure.
Since Blown rapeseed oil 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 25 hours.
Subsequently, 16 ml synthetic sewage feed, 200 ml sludge and Milli-RO water up to 500 ml were added resulting in a loading rate of 100 mg/l. Optimal contact between the test substance and test organisms was ensured applying continuous aeration and stirring during the 3-hour exposure time.

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 (4.0 g/l of sludge, as used for the test). The pH was 7.9 on the day of 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.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Post exposure observation period:
Oxygen consumption was measured and recorded for approximately 10 minutes after the 3 hours exposure period.

Test conditions

Test temperature:
Between 18.9 and 19.9°C.
pH:
Controls: 7.7
Blown rapeseed oil: 7.5 - 7.6
Dissolved oxygen:
Oxygen consumption:
Control series: 67-68 mg O2/l/h
Blown rapeseed oil series: 65-72 mg O2/l/h
Control series, the oxygen concentration at the start of the measurments was at least 6.8 mg/l
Blown rapeseed oil series: the oxygen concentration at the start of the measurments was 3.9 and 7.2 mg/l , for the duplicate measurements.

Nominal and measured concentrations:
Loading rate of 100 mg/l.
Details on test conditions:
TEST SYSTEM
- Type (delete if not applicable): 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):2
- No. of vessels per control (replicates):2/ series
- Biomass loading rate: 1.6 g/l suspended solids in final test mixture

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap-water purified by reverse osmosis (Milli-RO
EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : 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
- Test concentrations: limit study at 100 mg/l

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Milli-RO water (tap water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA).

OTHER TEST CONDITIONS
- Adjustment of pH: no
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol

Results and discussion

Effect concentrationsopen allclose all
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
100 other: mg/l (loading rate)
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 100 other: mg/l (loading rate)
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
No significant inhibition of respiration rate of the sludge was recorded at a loading rate of 100 mg Blown rapeseed oil per litre. The duplicate measurement confirmed the result of the first measurement. Therefore, no further testing was needed. Hence, the EC50 of Blown rapeseed oil exceeded a loading rate of 100 mg/l.

Results with reference substance (positive control):
Results with reference substance valid: yes.
The inhibitory effect of 3,5-dichlorophenol on aerobic waste water (activated sludge) bacteria increased with increased with increasing concentration , ranging from a 43 % inhibition at 5,0 mg/l to 87% at 30 mg/l.
Relevant effects levels: The EC50 of 3,5-dichlorophenol was: 6.0 mg/l.
Reported statistics and error estimates:
For the reference substance the percentage inhibition was plotted against the logarithm of the concentrations and the EC50 was determined using linear regression analysis.
For Blown rapeseed oil no EC50 could be calculated because the test substance proved to be non-toxic (EC50 > 100 mg/l).

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Blown rapeseed oil was not toxic to waste water (activated sludge) bacteria at a loading rate of 100 mg/l. The 3-hour EC50 of Blown rapeseed oil exceeded a loading rate of 100 mg/l.