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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental Starting Date - 09 June 2008; Experimental Completion Date - 12 June 2008; Study Completion Date - 28 Aug 2008.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Version / remarks:
1984
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Identity: FAT 66042/A TE
Batch No.: 0582900
Purity: >80 %
Expiration date: 25-Feb-2013
pH: 9.5 at concentration of 10 g/L
Aggregate state / physical form at room temperature: solid
Storage conditions: At room temperature at about 20 °C, away from direct sunlight.
Analytical monitoring:
no
Vehicle:
no
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Test organisms: The study was performed with aerobic activated sludge from a wastewater treatment plant (ARA Ergolz II, Fullinsdorf / Switzerland) treating predominantly domestic waste water.
- Pretreatment: 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 ratio of wet to dry weight, 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 wastewater* per litre 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 6.6.

* Synthetic wastewater, filled up to a final volume of 1 litre with deionized water:
16 g peptone
11 g meat extract
3 g urea
0.7 g NaCl
0.4 g CaCl2 x 2H2O
0.2 g MgSO4 x 7H2O
2.8 g K2HPO4
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Test temperature:
The temperature in the test media, measured in one control, was 20 °C at the start and at the end of the incubation period.
pH:
between 7.1-8.0.
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 8.1 mg/l.
Nominal and measured concentrations:
The final nominal concentration tested was 1000 mg/l.
Details on test conditions:
TEST SYSTEM
- Test vessel: 2000-ml glass beakers.
- Inoculum: Inoculum had a sludge concentration of 3.5 g/l dry weight (corresponding to about 1.4 g dry material per litre test medium). The sludge was added in time intervals of 15 minutes (an arbitrary but convenient interval) first to a control, secondly to the test solutions of the reference item, thirdly to the test solution of the test item, and finally to the second control.
- Aeration: During the incubation period of 3 hours the single test medium and the controls were continuously aerated by intense stirring on magnetic stirrers to avoid possible foaming and/or stripping of the test item.

TEST MEDIUM / WATER PARAMETERS
- Water: Synthetic wastewater, filled up to a final volume of 1 litre with deionized water:

EFFECT PARAMETERS MEASURED
- Measurement of the Respiration Rates: For measurement of the respiration rate a well-mixed sample of each test medium was poured into a BOD-flask after three hours incubation time, and was not further aerated. Then the dissolved oxygen concentration was measured with an oxygen electrode and was continuously recorded. During measurement, the samples were continuously stirred on a magnetic stirrer.
- Determination of the Inhibition Rates: The inhibitory effect of the test item or of the reference item on the respiration rate (oxygen consumption per minute) is expressed as a percentage of the mean respiration rate of the two controls measured at the start and at the end of the test series.

TEST CONCENTRATIONS
- Test concentrations: 500.4 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 dissolved in the tap water by ultrasonic treatment for fifteen minutes and intense stirring for ten minutes at room temperature. No emulsifiers or solvents were used. After stirring, the appearance of the test medium was evaluated, and it was a clear yellowish solution. Then, 16 ml synthetic wastewater and 200 ml activated sludge inoculum were added.
- Controls: two controls containing only tap water, synthetic wastewater and inoculum were tested in parallel to the single test concentration of the test item under identical test conditions.

PRE EXPERIMENT
Based on the results of a pre-experiment (without GLP), the test item was soluble at a concentration of at least 1000 mg/l in tap water.
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol, purity: 98.8 %
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
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
Details on results:
At the test item concentration of 1000 mg/l, the test item was completely dissolved in the test medium. The test item had no significant inhibitory effect (≤ 15 %) on the respiration rate of activated sludge after the incubation period of 3 hours at the limit test concentration of 1000 mg/l. Thus, the 3-hour NOEC (EC15) of to activated sludge microorganisms was at least 1000 mg/l. This value might even be higher but concentrations above 1000 mg/l were not tested. The 3-hour EC20, EC5O, and EC80 could not be calculated but were clearly higher than 1000 mg/l. The oxygen consumption rates of the two controls (run at the start and at the end of the test) differed by 9 % (guideline-recommended maximum variation: 15 %).
Results with reference substance (positive control):
The 3-hour EC50 of the reference item 3,5-dichlorophenol (positive control) was calculated to be 15 mg/l (the 95 % confidence limits could not be calculated). The 3-hour EC5O is within the guideline-recommended range of 5-30 mg/l, confirming suitability of the activated sludge used.

Influence of the Test Item and 3,5-Dichlorophenol (Reference Item) on the Oxygen Consumption of Activated Sludge

Vessel N. Nominal Conc. Of test chemical (mg/l) Oxygen consumption rate (mg O2/l min Inhibition pH values Oxygen conc. (mg O2/l)
Start* end* Start*WS end*c@
Control
A 0 1.331 7.2 7.9 8.4 8.1
B 0 1.213 7.2 8.0 7.5 8.7
Mean 1.272    
Deviation (%) 9

Test item

6 1000 1.300 -2.2 7.2 7.9 8.0 8.7

Reference item

1 5 1.188 6.7 7.1 7.9 8.4 8.7
2 16 0.552 56.6 7.1 8.0 8.5 8.6
3 50 0.148 88.4 7.1 8.0 8.5 8.6

* Start and end of the 3-hour incubation period

- % inhibition: increased oxygen consumption rate relative to control

Validity criteria fulfilled:
yes
Conclusions:
The 3-hour EC50 was calculated to be greater than 1000 mg/l.
Executive summary:

The inhibitory effect of the test item on the respiration rate of aerobic wastewater microorganisms of activated sludge was investigated in a 3-hour respiration inhibition test according to the ED Commission Directive 88/302/EEC, Part C.ll, and the OECD Guideline for Testing of Chemicals, No. 209. In accordance with the ED Commission Directive 92/69/EEC (C.1-C.3), 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. The test item had 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. Thus, the 3-hour NOEC (EC15) of the test item to activated sludge microorganisms was at least 1000 mg/l. 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.

Description of key information

The inhibitory effect of the test item on the respiration rate of aerobic wastewater microorganisms of activated sludge was investigated in a 3-hour respiration inhibition test according to the ED Commission Directive 88/302/EEC, Part C.ll, and the OECD Guideline for Testing of Chemicals, No. 209. In accordance with the ED Commission Directive 92/69/EEC (C.1-C.3), 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. The test item had 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. Thus, the 3-hour NOEC (EC15) of the test item to activated sludge microorganisms was at least 1000 mg/l. 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.

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L

Additional information