Registration Dossier

Administrative data

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2009-12-04 - 2010-01-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline-conform study under GLP, without deviations.

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
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): Tert-butyl-4-methoxy-4'-dibenzoylmethane
- Substance type: organic mono-constituent
- Physical state: solid (powder)

Sampling and analysis

Analytical monitoring:
not required
Details on sampling:
- Concentrations: 1000 mg/l
- Sampling method: not applicable
- Sample storage conditions before analysis: not applicable

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 500.1 mg of the test item was weighed by means of an analytical balance and transferred to the designated test vessel with 284 mL tap water. The test item was mixed into the tap water by ultrasonic treatment for fifteen minutes and intense stirring for 24 hours at room temperature in the dark to dissolve a maximum amount of the test item and/or disperse it as homogeneously as possible. No emulsifiers or solvents were used.
- Eluate: not applicable
- Differential loading: not applicable
- Controls: Two controls containing only tap water, synthetic sewage feed and inoculum were tested in parallel (inoculum control). The reference item 3,5-dichlorophenol was tested in parallel under identical test conditions, and functioned as a positive control.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): not applicable
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): not applicable
- Evidence of undissolved material (e.g. precipitate, surface film, etc): At the test item concentration of 1000 mg/L, the test item was not completely dissolved in the test medium. Thus, the test concentration was clearly above the water solubility limit of Tert-butyl-4-methoxy-4'-dibenzoylmethane under the test conditions.

Test organisms

Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Laboratory culture: The study was performed with aerobic activated sludge from a wastewater treatment plant (ARA Ergolz II, Füllinsdorf / Switzerland) treating predominantly domestic wastewater.
- Method of cultivation: The sludge was washed twice with tap water by centrifugation and the supernatant liquid phase was decanted. A homogenized aliquot of the final sludge suspension was weighed, thereafter dried and the ratio of wet to dry weight was calculated. An aliquot of washed sludge was suspended in tap water to obtain a concentration equivalent to 3 g dry material per liter. During the holding period of two days prior to use, the sludge was fed daily with 50 mL synthetic sewage feed per liter and was kept at room temperature under continuous aeration until use.
- Preparation of inoculum for exposure: Before use, the dry weight of the activated sludge was measured again in the inoculum used for the test. The pH of the activated sludge inoculum was 7.1. At the start of the test (after the stirring period of 24 hours), synthetic sewage feed and activated sludge inoculum were added.
- Initial biomass concentration: The inoculum had a sludge concentration of 2.1 g/L dry weight (corresponding to about 0.8 g dry material per liter test medium).

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Post exposure observation period:
not applicable

Test conditions

Hardness:
120 mmol/l
Test temperature:
20 °C
pH:
7.7
Dissolved oxygen:
The concentration of dissolved oxygen did not drop below 2.5 mg/L during the incubation period. Just before measurement of the respiration rates the dissolved oxygen concentrations were at least 8.0 mg/L
Salinity:
Not specified, however the detailed composition of the synthetic sewage feed medium is described in the study report.
Nominal and measured concentrations:
Nominal test item concentration: 1000 mg/l
Details on test conditions:
TEST SYSTEM
- Test vessel: beakers.
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: glass, 2000-mL size, 1500 mL head space, 500 mL fill volume
- Aeration: continuously aerated by intense stirring on magnetic stirrers
- Type of flow-through (e.g. peristaltic or proportional diluter): not applicable
- Renewal rate of test solution (frequency/flow rate): not applicable
- No. of organisms per vessel: not applicable
- 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: 2.1 g/L dry weight

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Synthetic sewage feed, filled up to a final volume of 1 liter with deionized water
- Total organic carbon: Not specified, however the detailed composition of the synthetic sewage feed medium is described in the study report.
- Particulate matter: Biomass loading 2.1 g/L dry weight
- Metals: Na, K, Mg, Ca
- Pesticides: not applicable
- Chlorine: not applicable
- Alkalinity: Not specified, however the detailed composition of the synthetic sewage feed medium is described in the study report.
- Ca/Mg ratio: 0.44
- Conductivity: Not specified, however the detailed composition of the synthetic sewage feed medium is described in the study report.
- Culture medium different from test medium: no
- Intervals of water quality measurement: The pH values and dissolved oxygen concentrations were determined after the addition of synthetic sewage feed and activated sludge in all test media and the controls at the start and at the end of the 3-hour incubation period. The water temperature was measured in one control at the start and at the end of the incubation period. Before the addition of activated sludge and synthetic sewage feed, the appearance of the test media was recorded.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: test solutions were kept in the dark
- Light intensity: not applicable

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : dissolved oxygen consupmtion in test solutions

TEST CONCENTRATIONS
- Spacing factor for test concentrations: not applicable
- Justification for using less concentrations than requested by guideline: Based on the EU testing guidelines, a limit test with a nominal test concentration of 1000 mg/L was performed.
- Range finding study: not reported
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol

Results and discussion

Effect concentrationsopen allclose all
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Duration:
3 h
Dose descriptor:
other: EC20
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Duration:
3 h
Dose descriptor:
other: EC80
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
>= 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
>= 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Based on the physical appearance of the test suspension, the saturation concentration (water solubility limit of Tert-butyl-4-methoxy-4'-dibenzoylmethane under the test conditions) was <1000 mg/L and resulted in less than 15% inhibition after three hours contact time.
- Effect concentrations exceeding solubility of substance in test medium: see above
- EC50, EC80, and EC20 values could not be calculated, but were clearly higher than 1000 mg/L.
- The 3-hour NOEC might even be higher but concentrations above 1000 mg/L were not tested.

Results with reference substance (positive control):
- Results with reference substance valid? Yes. The 3-hour EC50 is within the guideline-recommended range of 530 mg/L, confirming suitability of the activated sludge used.
- Relevant effect levels: The 3-hour EC50 of the reference item 3,5-dichlorophenol (positive control) was calculated to be 12 mg/L
- Other:
Reported statistics and error estimates:
No statistics applied.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The 3-hour NOEC (EC10) of the test item to activated sludge microorganisms was at least 1000 mg/L (i.e. the saturation concentration of the test item). This value might even be higher but concentrations above 1000 mg/L were not tested. The 3-hour EC20, EC50, and EC80 could not be calculated but were clearly higher than 1000 mg/L.
Executive summary:

The inhibitory effect of the test item Tert-butyl-4-methoxy-4-dibenzoylmethane on the respiration rate of aerobic wastewater microorganisms of activated sludge was investigated in a 3-hour respiration inhibition test according to OECD Guideline 209 and EU Commission Directive 88/302/EEC, Part C.11. Based on the EU testing guidelines, a limit test with a nominal test concentration of 1000 mg/L was performed. In addition, two controls and three different concentrations of the reference item 3,5-dichlorophenol (5, 16, and 50 mg/L) were tested in parallel. The results of these treatments confirmed suitability of the activated sludge and the method used. At the test item concentration of 1000 mg/L, the test item was not completely dissolved in the test medium. Thus, the test concentration was clearly above the water solubility limit of Tert-butyl-4-methoxy-4-dibenzoylmethane under the test conditions. The test item had no inhibitory effect on the respiration rate of activated sludge after the incubation period of three hours at a loading rate of 1000 mg/L. The saturation concentration (water solubility limit of Tert-butyl-4-methoxy-4-dibenzoylmethane under the test conditions) was reached and no inhibitory effect was determined after three hours contact time. Thus, the 3-hour NOEC (EC10) of the test item to activated sludge microorganisms was at least 1000 mg/L (i.e. the saturation concentration of the test item). This value might even be higher but concentrations above 1000 mg/L were not tested. The 3-hour EC20, EC50, and EC80 could not be calculated but were clearly higher than 1000 mg/L.