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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 February 2011 to 02 March 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant, guideline study, no restrictions, fully adequate for assessment.
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)
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Not applicable
Analytical monitoring:
no
Details on sampling:
Not applicable
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: No stock solution was made due to the low water solubility of the test item. 500.0mg of test substance was weighed by means of an analytical balance and transferred to the designated test vessels with 284 mL tap water. The test item was mixed into the tap water by ultrasonic treatment for fifteen minutes and intense stirring for three hours at room temperature in the dark to dissolve a maximum amount of the test item and/or disperse it as homogeneously as possible.
- Eluate: Not applicable
- Controls: Two blank controls of tap water containing synthetic sewage feed and inoculum. Reference item 3, 5-dichlorophenol was tested in parallel at three concentrations under identical conditions as a toxicity control.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): Appearance of test media showed that the single test concentration was clearly above the water solubility limit of the test item.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Laboratory culture: No
- Method of cultivation: Aerobic activated sludge from a wastewater treatment plant (ARA Ergolz II, Füllinsdorf / Switzerland) treating predominantly domestic wastewater was collected. 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:
- Preparation of inoculum for exposure: Based on the wet to dry weight ratio, an aliquot of washed sludge was suspended in tap water to obtain a concentration equivalent to 3 g dry material per litre. During the holding period of three 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. 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 7.2.
* Synthetic sewage feed:
16 g peptone
11 g meat extract
3 g urea
0.7 g NaCl filled up to a final volume of 1 liter
0.4 g CaCl2 × 2H2O with deionized water
0.2 g MgSO4 × 7H2O
2.8 g K2HPO4
Made up to 1 litre with deionized water.

- Pretreatment: None
- Initial biomass concentration: 2.2 g/L dry weight (corresponding to about 0.9 g dry material per litre test medium).
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Post exposure observation period:
None reported
Hardness:
Not reported
Test temperature:
19ºC to 20ºC
pH:
7.3 to 8.6
Dissolved oxygen:
7.6 mg/L to 8.6 mg/L after 3-hour incubation period and just before respiration rate measurements
Salinity:
Not applicable
Nominal and measured concentrations:
Test substance nominal concentrations: 1000 mg/L.
Reference substance nominal concentrations: 5, 16 and 50 mg 3, 5-dichlorophenol/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Open
- Material, size, headspace, fill volume: 2000 mL glass beakers filled with 500 mL solution.
- Aeration: Continuous aeration by intense stirring with magnetic stirrer.
- Sludge concentration: 2.2 g/L dry weight (corresponding to about 0.9 g dry material per litre of test medium).
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 2
- No. of vessels per reference item(replicates): 1

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: tap water
- Total organic carbon: Not reported
- Particulate matter: Not reported
- Metals: Not reported
- Pesticides: Not reported
- Chlorine: Not reported
- Alkalinity: Not reported
- Ca/mg ratio: Not reported
- Conductivity: Not reported
- Culture medium different from test medium: No.
- Intervals of water quality measurement: Not reported

OTHER TEST CONDITIONS
- Adjustment of pH: None


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Oxygen respiration rate measured continuously after 3 hour incubation period (see below for more details).

Reference substance (positive control):
yes
Remarks:
3, 5-Dichlorophenol
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
Remarks on result:
other: Limit test.
Details on results:
- Effect concentrations exceeding solubility of substance in test medium: 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 the test item under the test conditions.

3-hour EC20, EC50 and EC80 could not be determined but where clearly higher than 1000 mg CA4920/L.



Results with reference substance (positive control):
- Results with reference substance valid? Yes
- Relevant effect levels: The 3-hour EC50 of the reference item 3,5-dichlorophenol (positive control) was calculated to be 9 mg/L (the 95% confidence limits could not be calculated due to mathematical reasons). The 3-hour EC50 is within the guideline-recommended range of 5–30 mg/L, confirming suitability of the activated sludge used.
Reported statistics and error estimates:
The 3-hour EC50 value of the reference item 3, 5-dichlorophenol was calculated by Probit Analysis

Table 1. Influence of Test Item and 3, 5-Dichlorophenol (reference item) on the Oxygen Consumption of Activated Sludge
Vessel no. Nominal concentration of test chemical (mg/L) Oxygen consumption rate (mg O2L-1min-1) Inhibition (%) pH values Oxygen Concentration (mg O2/L
start* end* start* end*
Control
A 0 1.033 7.3 8.5 8.1 7.9
B 0 1.078 7.5 8.4 8.0 8.0
Mean  1.056
Deviation (%) 4.3
Test Item
5 1000 1.056 -0.1 7.6 8.4 8.3 8.2
Reference item
1 5 0.821 22.2 7.6 8.6 8.1 7.6
2 16 0.24 77.3 7.5 8.6 8.2 8.6
3 50 0.089 91.6 7.5 8.6 8.1 8.6
* start and end of the 3-hour incubation period
-% inhibition: increased oxygen consumption rate relative to control
Validity criteria fulfilled:
yes
Remarks:
The oxygen consumption rates of the two controls is within 15% of each other and the 3-hour EC50 of the reference substance is within the guideline-recommended range of 5 - 30 mg 3, 5-dichlorophenol/L.
Conclusions:
In a limit test, the 3-hour NOEC value for the inhibitory effect of the test material on the respiration rate of aerobic activated sludge microorganisms was 1000 mg/L, based on nominal concentrations. The test concentrations was clearly above the water solubility of the test material under the test conditions. The test item has no inhibitory effect ( ≤ 10%) on the respiration rate of activated sludge after the incubation period of three hours at the test item concentration of 1000 mg/L.
Executive summary:

The sensitivity of activated sludge microorganisms to the test material was determined in a GLP-compliant test performed to standard guidelines. In a limit test, the test item has no inhibitory effect on the respiration rate of activated sludge after the incubation period of three hours at the test item concentration of 1000 mg/L.. The 3-hour NOEC value was determined to be 1000 mg/L. The results are based on nominal concentrations and the test concentration was clearly above the water solubility of the test material under the test conditions.

Description of key information

In a limit test, the 3-hour NOEC  value for the inhibitory effect of the test material on the respiration rate of aerobic activated sludge microorganisms was  1000 mg/L, based on nominal concentrations.  The test concentrations was clearly above the water solubility of the test material under the test conditions.  The test item has no inhibitory effect ( ≤ 10%) on the respiration rate of activated sludge after the incubation period of three hours at the test item concentration of 1000 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
1 000 mg/L

Additional information

The sensitivity of activated sludge microorganisms to the test material was determined in a GLP-compliant test performed to standard guidelines. In a limit test, the test item has no inhibitory effect on the respiration rate of activated sludge after the incubation period of three hours at the test item concentration of 1000 mg/L.. The 3-hour NOEC value was determined to be 1000 mg/L. The results are based on nominal concentrations and the test concentration was clearly above the water solubility of the test material under the test conditions.