Registration Dossier

Administrative data

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to OECD TG 209 and EU Guideline C.11 and in accordance with the Principles of Good Laboratory Practice (GLP).
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

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
Remarks:
exceptions, eg. characterization and stability of reference substance not determined according to GLP, concentration, stability and homogeneity of both reference and test substance were not determined, however overall outcome or integrity not affected
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
no
Remarks:
same as above
Principles of method if other than guideline:
not applicable
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): 1,4,5,8-Tetra (4'-n-butylphenylamino) Anthraquinone (TBPAAQ)
- Substance type: green powder
- Physical state: solid
- Analytical purity: > 96% active ingredient
- Lot/batch No.: Lot #20070406; TD No. 07-016
- Storage condition of test material: room temperature
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
not applicable

Sampling and analysis

Analytical monitoring:
no
Details on sampling:
- Concentrations: 0.1,0.3 and 0.5 µg/L

Test solutions

Vehicle:
yes
Details on test solutions:
A stock solution of the test substance was prepared at a nominal concentration of 10 mg/L in Tetrahydrohran. The appropriate amount of stock solution for each test vessel was applied to a glass fiber filter, the solvent was allowed to evaporate, and then each filter was added to the appropriate test vessel. This method was deemed appropriate based on the solubility of the test substance in Tetrahydrofuran.

Test organisms

Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
Activated sludge was collected from the Denton Wastewater Treatment Plant, Denton, Maryland and was utilized as the inoculum for the test. The Denton facility receives wastes from predominately domestic sources. The sludge was sieved using a 2 mm screen and allowed to settle for approximately 30 minutes. The supernatant above the settled solids was removed and the total suspended solids (TSS) concentration of the settled sludge was determined. Total suspended solids in the settled sludge were adjusted to a nominal concentration of approximately 4000 mg/L by dilution with municipal water. Fifty mL of synthetic sewage was added to each liter of adjusted sludge. The sludge was maintained at a temperature of 20 ± 2°C and continuously aerated overnight. Before use, the pH and total suspended solids concentration of the activated sludge were determined.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Post exposure observation period:
not applicable

Test conditions

Hardness:
not applicable
Test temperature:
Temperatures measured throughout the period of sludge maintenance and during the test ranged from 20.1-21.3 °C.
pH:
The measured initial pH of the sludge on the day of testing was 8.0 prior to use in the test system.
Dissolved oxygen:
not applicable
Salinity:
not applicable
Nominal and measured concentrations:
Nominal concentrations: 0.1,0.3 and 0.5 µg/L
Details on test conditions:
A stock solution of the reference substance, 3,5-dichlorophenol, was prepared by dissolving 500.1 mg of the reference substance in 10 mL of 1N NaOH and then diluting to 30 mL with pure water. While stirring, 9.4 mL of 1N H2S04 was added to reach the point of incipient precipitation. The solution of 3,5-dichlorophenol was then diluted to a final volume of 1L with pure water. The reference substance solution was administered to the reference group by volumetric addition.

Control, solvent control, reference, and treatment test mixtures were incubated at 20 ± 2 °C and aerated for three hours at a rate sufficient to provide aerobic conditions and maintain solids in suspension. The mixtures were prepared and aerated in 1000 mL Erlenmeyer flasks and then transferred to fill 300 mL biochemical oxygen demand (BOD) bottles to conduct dissolved oxygen (DO) measurements. Test mixtures were identified by project number, test substance identification, test concentration, and bottle number.

Test mixtures were prepared at 15 minute intervals starting with the first control. The controls and solvent control contained 16.0 mL of synthetic sewage, 200 mL of inoculum, and enough municipal water to bring the total volume up to 500 mL. The mixtures were promptly aerated at a rate sufficient to provide aerobic conditions and keep the solids in suspension. Subsequent mixtures contained 16.0 mL of synthetic sewage, 200 mL of inoculum, the appropriate amount of test substance stock solution applied to a glass fiber filter or reference substance stock solution, and enough municipal water to bring the total volume up to 500 mL. Finally, a second control was prepared. All mixtures were aerated for three hours.

The contents of the first control vessel were transferred to a BOD bottle and dissolved oxygen concentrations were measured every 10 seconds over a 10 minute period, or until dissolved oxygen concentrations fell below 1.0 mg/L, using a YSI Model 50B Dissolved Oxygen Meter. The respiration rate in subsequent vessels was determined in an identical manner at 15 minute intervals. The contact time for each concentration of reference or test substance with the activated sludge was three hours.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol (Lot #15809KI, purity 97%)

Results and discussion

Effect concentrations
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 0.5 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: highest concentration tested
Details on results:
Temperatures measured throughout the period of sludge maintenance and during the test ranged from 20.1-21.3 °C. The initial total suspended solids (TSS) concentration of the activated sludge after removal of the supernatant layer measured 8947 mg/L. The measured TSS concentration of the sludge on the day of testing was 4146.7 mg/L. The measured initial pH of the sludge on the day of testing was 8.0 prior to use in the test system.

The respiration rates observed in the two controls were 30.6 and 35.5 mg 02/Lihr, with a difference of approximately 9%.

The observed percent inhibitions for 1,4,5,8-Tetra (4'-n-butylphenylamino) Anthraquinone (TBPAAQ) ranged from -9.5 to -2.0% and were not concentration dependent.
Results with reference substance (positive control):
The EC50 value for the reference substance was 19.4 mg/L with 95% confidence limits of 3 mg/L and 50 mg/L and was within the 5 to 30 mg/L range considered acceptable for the test
Reported statistics and error estimates:
Standard statistical methods were employed. The EC50 and 95% confidence limits were calculated using binomial probability with nonlinear interpolation.

Any other information on results incl. tables

None

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Inhibitory effects upon respiration by 1,4,5,8-Tetra (4'-n-butylphenylamino) Anthraquinone (TBPAAQ) at the concentrations evaluated in this study were variable and did not exhibit a concentration dependent dose response pattern. The EC50 value for 1,4,5,8-Tetra (4'-nbutylphenylamino) Anthraquinone (TBPAAQ) is therefore greater than 0.5 µg/L, the highest concentration tested.
Executive summary:

The effect of 1, 4, 5, 8-Tetra (4'-n-butylphenylamino) Anthraquinone (TBPAAQ) on activated sludge microorganisms maintained in an aerobic environment was assessed by the Activated Sludge Respiration Inhibition Test Method (OECD Guideline 209).

The test contained control, reference and treatment groups and a solvent control. The control replicates were used to determine the background respiration rate of the sludge and were not dosed with the test or reference substance. The reference group was dosed with 3, 5- dichlorophenol, a known inhibitor of respiration, at concentrations of 3, 15 and 50 mg/L. The treatment group was dosed with 1,4,5,8-Tetra (4'-n-butylphenylamino) Anthraquinone (TBPAAQ) at concentrations of 0.1, 0.3 and 0.5 μg/L. The solvent control included a glass fiber filter dosed with the maximum amount of solvent used for a test vessel in the study. After an exposure period of three hours, the respiration rates of the solutions were measured using a YSI Model 50 B Dissolved Oxygen Meter.

The respiration rates in the controls were 30.6 and 33.5 mg 02/L/hr with a difference of approximately 9%, and were within the 15% difference limit established for the test. The validity of the test was further supported by the results from the 3,5-dichlorophenol reference group. The EC50 value of the reference substance was calculated using nonlinear interpolation between 15 and 50 mg/L and was found to be 19.4 mg/L with 95% confidence limits of 3 mg/L and 50 mg/L at a contact time of 3.0 hours. A dose response effect was not observed for the treatment group. The EC50 value for 1,4,5,8-Tetra (4'-nbutylphenylamino) Anthraquinone (TBPAAQ) is therefore greater than 0.5 μg/L, the highest concentration tested.