Registration Dossier

Administrative data

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2006-10-12 to 2007-06-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study performed according to OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test) and EC Directive 88/302, ‘Biodegradation - Activated Sludge Respiration Inhibition Test’ without deviations.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
Remarks:
: The testing facility indicated that there were no deviations from the protocol.
Qualifier:
according to
Guideline:
other: EC Directive 88/302, ‘Biodegradation - Activated Sludge Respiration Inhibition Test’
Deviations:
no
Remarks:
: The testing facility indicated that there were no deviations from the protocol.
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
: Certificate issued by the United Kingdom Department of Health.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): VRT-753136
- Molecular formula (if other than submission substance): Not applicable
- Molecular weight (if other than submission substance): Not applicable
- Smiles notation (if other than submission substance): Not applicable
- InChl (if other than submission substance): Not applicable
- Structural formula attached as image file (if other than submission substance): Not applicable
- Substance type: No data
- Physical state: White powder
- Analytical purity: 99.4% w/w by High Performance Liquid Chromatography (HPLC)
- Impurities (identity and concentrations): individual unknown components were detected at 0.03 area%; isopropanol (residual solvent) at 300 ppm
- Composition of test material, percentage of components: Not applicable
- Isomers composition: Not applicable
- Purity test date: 2006-05-25
- Lot/batch No.: 25515
- Expiration date of the lot/batch: 2008-05-01
- Stability under test conditions: No data
- Storage condition of test material: Room temperature
- Other: No data
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:
not specified
Details on sampling:
Not applicable

Test solutions

Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION:
- Method: The result of preliminary solubility trials showed that the test substance was sufficiently soluble in dechlorinated water to allow the preparation of suitable aqueous stock solutions. Therefore, test concentrations were established by the addition of appropriate volumes of stock solutions at 2.5 g/L in the preliminary test and 16 g/L in the definitive test. The pH of each stock solution was measured and no adjustment was necessary. Additions of synthetic sewage and microbial inoculum, made at fifteen-minute intervals, gave a final volume of 500 mL.
- Eluate: not applicable
- Differential loading: not applicable
- Controls: yes (16 mL synthetic sewage + 284 mL water + 200 mL inoculum)
- Chemical name of vehicle: dechlorinated tap water (hardness, 200-250 mg/L as CaCO3)
- Concentration of vehicle in test medium: 34-268.4 mL per 500 mL final volume
- Evidence of undissolved material: no data

Test organisms

Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Laboratory culture: Not applicable
- Method of cultivation: No data
- Preparation of inoculum for exposure: On the day of collection, aliquots (25 mL) of the activated sludge were filtered through dried and preweighed Whatman GF/C filter papers which were then dried again at approximately 105 deg C for at least one hour, allowed to cool in a desiccator and reweighed. The mixed liquor suspended solids (MLSS) content of the activated sludge was then calculated and adjusted to 4 g/L by the removal of supernatant (in the preliminary test only). Synthetic sewage (50 mL/L) was added to each stock of activated sludge and these were aerated overnight. On the day of the test, the MLSS content of the sludge was determined and adjusted to 4 g/L by the addition of dechlorinated tap water. The pH and temperature of the sludge were also measured.
- Pretreatment: No
- Initial biomass concentration: 4 g/L

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Post exposure observation period:
None

Test conditions

Hardness:
200-250 mg/L CaCO3
Test temperature:
- Control test: 20 deg C
- Preliminary test: 20+-2 deg C
- Definitive test: 19.9+-2 deg C
pH:
- Control test: 7.6-8.1
- Preliminary test: 7.5-8.3
- Definitive test: 7.4-8.1
Dissolved oxygen:
- Control test: 6.1 mg O2/L (t=0); 2.5 mg O2/L (t=3h)
- Preliminary test: 6.5 mg O2/L (t=0); 2.5 mg O2/L (t=3h)
- Definitive test: 6.5-7.2 mg O2/L (T0) 2.5-4 mg O2/L (t=3h)
Salinity:
No data
Nominal and measured concentrations:
- Preleminary test: 10, 100, 1000, 1000, 1000 mg/L (Nominal concentration)
- Definitive test: 500, 1000, 2000, 4000 and 8000 mg/L (Nominal concentrations)
- Positive control: 3, 10 and 32 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type: Thermostatically controlled water bath
- Material, size, headspace, fill volume: No data
- Aeration: Yes, using a glass aerator connected to a laboratory supply of oil-free compressed air (ca. 1 L/min)
- Type of flow-through: Not applicable
- Renewal rate of test solution: Not applicable
- No. of organisms per vessel: No data
- No. of vessels per concentration (replicates): No data
- No. of vessels per control (replicates): No data
- No. of vessels per vehicle control (replicates): No data
- Biomass loading rate: No data

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: tap water that had been softened and treated by reverse osmosis and then purified to give a nominal resistivity >= 18 MegOhm.cm
- Total organic carbon: No data
- Particulate matter: No data
- Metals: No data
- Pesticides: No data
- Chlorine: No data
- Alkalinity: No data
- Ca/mg ratio: No data
- Conductivity: No data
- Culture medium different from test medium: No data
- Intervals of water quality measurement: No data

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: No data
- Light intensity: No data

EFFECT PARAMETERS MEASURED:
- The rate of oxygen consumption was measured, over a period of approximately 10 minutes or until the dissolved oxygen concentration fell below 2 mgO2/L.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: No data
- Justification for using less concentrations than requested by guideline: Not applicable
- Range finding study: Yes
- Test concentrations: Nominal test concentrations were 0, 100 and 1000 mg/L (highest concentration tested in triplicate).
- Results used to determine the conditions for the definitive study: The results of preliminary solubility trials showed that the test substance was sufficiently soluble in dechlorinated water to allow the preparation of suitable aqueous stock solutions.
Reference substance (positive control):
yes
Remarks:
: 3,5-dichlorophenol (3,5-DCP), 99.2%

Results and discussion

Effect concentrationsopen allclose all
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
9 690 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: 95% CI: 6820-16,000 mg/L
Duration:
3 h
Dose descriptor:
other: EC20
Effect conc.:
356 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: 95% CI: 171-614 mg/L
Details on results:
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No data
- Effect concentrations exceeding solubility of substance in test medium: No
Results with reference substance (positive control):
- Results with reference substance valid: Yes. Sludge respiration rates were progressively reduced in the presence of increasing concentrations of 3,5-DCP.
- Relevant effect levels: The 3-h EC50 for 3,5-DCP was calculated to be 11.6 mg/L (95% confidence limits, 9.1 - 14.9 mg/L) in the preliminary test, and 9.3 mg/L (95% confidence limits, 7.8 - 11.0 mg/L) in the definitive test. The specific respiration rates of control cultures established at the end of the two test series (26.3 and 24.6 mgO2/g/h) were 107% and 98% of the rates of those established at the start (24.6 and 25.0 mgO2/g/h). These results show that the tests were valid and that the sample of activated sludge employed was sensitive to inhibition.
- Other: no data
Reported statistics and error estimates:
The EC50 and 95% confidence limits of the test and reference substances were calculated using the SAFEstat curvefit program. Since the permitted maximum difference for specific respiration rates in control beakers established at the beginning and end of the test period was 15%, this value was used to define the criteria for biologically significant levels of inhibition in mixtures containing the test substance.

Any other information on results incl. tables

Table 1. Temperature, pH and respiration rate measurements.

Test mixture 

Initial Temperature  Final Temperature  Initial pH  Final pH  Specific respiration rate mgO2/g/h  % inhibition 

Control (1) 

19.9 

19.8  7.6  8.1  25.0  -  

Control (2) 

19.7 

19.7  7.6  8.1  24.6 

Test substance 

 

         

500 

19.8  19.6  7.5  8.1  19.0  23 

1000 

19.8  19.7  7.5  8.1  18.3  26 

2000 

19.9  19.6  7.4  7.9  16.3  34 

4000 

19.8  19.6  7.3  7.8  14.5  42 

8000 

19.8  19.7  7.1  7.6  13.0  47 

Positive control 

 

         

19.8  19.7  7.6  8.2  17.6  29 

10 

19.8  19.7  7.5  8.1  12.5  50 

32 

19.7  19.6  7.6  8.1  6.0  76 

Applicant's summary and conclusion

Conclusions:
Concentration-related inhibition of respiration rates occurred at each test substance concentration in the definitive test. The highest tested concentration of the test substance in the definitive test (8000 mg/L) caused 47% inhibition. The EC50 of the test substance was calculated from the results of the definitive test to be 960 mg/L (95% confidence limits, 6820 - 16,000 mg/L). The EC20 value was calculated to be 356 mg/L (95% confidence limits, 171 - 614 mg/L). The highest test concentration tested (8000 mg/L) caused 47% inhibition.
Executive summary:

Not applicable