Registration Dossier

Administrative data

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1 April to 4 April 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted with GLP compliance. Study report contains a GLP statement of compliance and GLP certificate

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report Date:
1997

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:

- Physical state: solid
- Analytical purity: 97.8%
- Impurities (identity and concentrations): No information
- Composition of test material, percentage of components: No information
- Isomers composition: No information
- Lot/batch No.: A105333
- Expiration date of the lot/batch: 18 March 1998
- Stability under test conditions: Stable in water for 48 hours (ref HRC study no ORN 30(b)/88726)
- Storage condition of test material: At room temperature at about 20C away from direct sunlight
- Other:
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
None

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: Not specified.
- Sampling method: After exactly 3 hours of aeration at 21 deg c, the first test flask (innoculum control flask 1) was taken for measurement of the oxygen concentration over a period of 8 minutes. Thereafter, the oxygen consumption was measured in the rest of the test flasks (Flasks 2-13) over a period of 7-9 minutes in 10 minute intervals.

Test solutions

Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: First optimally suspended in 5ml of dichloromethane (analytical grade quality) evaporated to dryness with a continuous stream of nitrogen and 248 ml of bidistilled water was added to each flask
- Treatment into solution: Ultrasonic processor (Vibra-Cell VCX-600, Sonics and Materials, Inc) under cooling with ice for 2 minutes
- Stirring: Stirred on magnetic stirrers for 24 hours at room temperature in the dark to reach saturation
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): 99.5, 49.9, 25.1, 12.6, 6.2 and 3.4 mg/l

Test organisms

Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Laboratory culture: Synthetic sewage feed
16g peptone
11g meat extract
3g urea
0.7g NaCl
0.4g CaCl2 . 2 H2O
0.2g MgSO4 . 7 H2O
2.8g K2HPO4

Filled up to 1 L with bidistilled water.

- Preparation of inoculum for exposure: 50ml of sludge feed per litre was added daily, starting 3 days prior to prior to use
- Storage prior to use: Kept at room temperature under constant aeration until use.
- Initial biomass concentration: 2.8 g/l
- pH: 8.0

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Post exposure observation period:
No information

Test conditions

Hardness:
No information
Test temperature:
21C
pH:
8.2 - 8.5
Dissolved oxygen:
No information
Salinity:
No information
Nominal and measured concentrations:
199, 100, 50, 25, 17 and 7 mg/l
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 1000 ml brown flask
- Aeration: Yes, immediately after filling the flasks, aeration using compressed air at an air-flow rate of approximately 0.5-1.0 litres per minute.
- Type of flow-through (e.g. peristaltic or proportional diluter): N/A
- Renewal rate of test solution (frequency/flow rate): N/A
- No. of organisms per vessel: N/A
- No. of vessels per concentration (replicates): No information
- No. of vessels per control (replicates): No information
- No. of vessels per vehicle control (replicates): No information


TEST MEDIUM / WATER PARAMETERS: No information


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


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : N/A


TEST CONCENTRATIONS: No information
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol

Results and discussion

Effect concentrations
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 199 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: oxygen consumption
Details on results:
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: 50, 25, 12 and 7 mg/l, white homogeneous suspension
- Effect concentrations exceeding solubility of substance in test medium: 199 and 100 mg/l

In comparison to the inoculums controls the respiration rate of the activated sludge was slightly inhibited by 11.4 and 10.6% at the two lowest nominal test concentrations of 7 and 12 mg/l respectively. At the next higher nominal test concentrations of 25, 50, 100 and 199 mg/l, the respiration rate reached a plateau with moderate inhibition rates of 35.0, 37.7, 38.9 and 32.3% respectively. These stagnant values are very likely a result of the attainment of a saturation equilibrium (limit of water solubility) under present test conditions. The test substance concentrations exceeding 199 mg/l nominal were not tested since they would have been above the water solubility given by the sponsor; moreover it is to be noted that for many low water soluble substances, the water solubility may vary greatly depending on the method used for the determination. From the appearance of the test substance in the final test medium, an obvious excess of test article was present at 100 and 100 mg/l. At the next lower concentrations of 50, 25, 12 and 7 mg/l, white homogenous suspensions were noted but a differentiation between a true solution and a colloidal dispersion could not be made by visual assessment.
From the inhibition rates, the 3-hour EC50 could not be quantified because the saturation concentration (equilibrium) under the present test conditions was reached, and resulted in less than 50% inhibition after three hours contact time. Nevertheless, the 3-hour EC50 is clearly higher than the limit of water solubility under the present test conditions, or >199 mg/l nominal. The 3 hour EC20 was calculated by linear regression analysis using the inhibition rates of the 12 and 25 mg/l test concentrations and was calculated to be 17.0 mg/l nominal.
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- Relevant effect levels: 5 - 30 mg/l
- Other:
The following nominal concentration of the positive control were tested on the same activated sludge and under identical conditions as the test article:
100, 32, 10 and 3.2 mg/l.
In comparison to the controls, the respiration rate of the activated sludge was not inhibited (-9.4%) at the lowest nominal concentration of 3.2 mg/l. At the next higher nominal concentrations of 10, 32 and 100 mg/l, the respiration rate was inhibited by 34.9, 80.6 and 91.6% respectively.
From these inhibition rates, the 3-hour EC50 was calculated by the Logit model to be 14.7 mg/l with the 95% confidence limits of 13.7 – 15.8 mg/l.
Reported statistics and error estimates:
none reported

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The 3-hour EC50 was considered to be higher than the water solubility limit of 199 mg/l nominal. The test sample is considered not to inhibit ready biodegradation by inhibiting the activity of activated sewage sludge.
Executive summary:

The acute toxicity of the test substance to aerobic waste water bacteria was measured to consider whether the test substance would inhibit the potential to readily biodegrade. Under the conditions of the test, the test substance is considered not to be toxic to bacteria in activated sewage sludge at the limit of the water solubility.