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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Version / remarks:
2010
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
NYMPHEAL (GR-88-0778) :
Lot No. : Batch 6
Purity : 99.1% (sum of the isomers)
Date of Expiry : 29 March, 2017
Analytical monitoring:
no
Remarks:
A dissolved oxygen measuring device composed of a flat bottom flask (BOD type, Karlsruher Flasche) in which an O2-electrode (StirrOx G, WTW, Weilheim, Germany) fits tightly and an oxygen-meter (inoLab Oxi 730, WTW, Weilheim, Germany).
Vehicle:
no
Details on test solutions:
Test Procedure :

Control C1: at time "0 minute", in a 500-ml scaled cylinder, 16 ml of the synthetic sewage feed was diluted to 250 ml with water. 250 ml of sludge were added and the mixture was aerated continuously for 3 hours by bubbling air into it.

Control C2: at time "15 minutes", in a 500-ml scaled cylinder, 16 ml of the synthetic sewage feed was diluted to 250 ml with water. 250 ml of sludge were added and the mixture was aerated continuously for 3 hours by bubbling air into it.

Control C3: at time "30 minutes", in a 500-ml scaled cylinder, 16 ml of the synthetic sewage feed was diluted to 250 ml with water. 250 ml of sludge were added and the mixture was aerated continuously for 3 hours by bubbling air into it.

Test S1: at time "45 minutes", in another 500-ml cylinder, 16 ml of the synthetic sewage feed + 50 mg of test substance were diluted to 250 ml with water. 250 ml of sludge were added and the mixture was aerated. Final test substance concentration: 100 mg/l.

Test S2: at time "60 minutes", in another 500-ml cylinder, 16 ml of the synthetic sewage feed + 50 mg of test substance were diluted to 250 ml with water. 250 ml of sludge were added and the mixture was aerated. Final test substance concentration: 100 mg/l.

Test S3: at time "75 minutes", in another 500-ml cylinder, 16 ml of the synthetic sewage feed + 50 mg of test substance were diluted to 250 ml with water. 250 ml of sludge were added and the mixture was aerated. Final test substance concentration: 100 mg/l.


Test R1: at time "90 minutes", in another 500-ml cylinder, 16 ml of the synthetic sewage feed + 1 mg of 3,5-dichlorophenol were diluted to 250 ml with water. 250 ml of sludge were added and the mixture was aerated. Final reference substance concentration:
2.00 mg/l

Test R2: at time "105 minutes", in another 500-ml cylinder, 16 ml of the synthetic sewage feed + 1 mg of 3,5-dichlorophenol were diluted to 250 ml with water. 250 ml of sludge were added and the mixture was aerated. Final reference substance concentration: 2.00 mg/L.

Test R3: at time "120 minutes", in another 500-ml cylinder, 16 ml of the synthetic sewage feed + 1 mg of 3,5-dichlorophenol were diluted to 250 ml with water. 250 ml of sludge were added and the mixture was aerated. Final reference substance concentration: 2.00 mg/l

Test R4: at time "135 minutes", in another 500-ml cylinder, 16 ml of the synthetic sewage feed + 12.5 mg of 3,5-dichlorophenol were diluted to 250 ml with water. 250 ml of sludge were added and the mixture was aerated. Final reference substance concentration: 25.00 mg/l

Test R5: at time "150 minutes", in another 500-ml cylinder, 16 ml of the synthetic sewage feed + 12.5 mg of 3,5-dichlorophenol were diluted to 250 ml with water. 250 ml of sludge were added and the mixture was aerated. Final reference substance concentration: 25.00 mg/l

Test R6: at time "165 minutes", in another 500-ml cylinder, 16 ml of the synthetic sewage feed + 12.5 mg of 3,5-dichlorophenol were diluted to 250 ml with water. 250 ml of sludge were added and the mixture was aerated. Final reference substance concentration: 25.00 mg/l

Control C4: at time "180 minutes", in a 500-ml scaled cylinder, 16 ml of the synthetic sewage feed was diluted to 250 ml with water. 250 ml of sludge were added and the mixture was aerated continuously for 3 hours by bubbling air into it.

Control C5: at time "195 minutes", in a 500-ml scaled cylinder, 16 ml of the synthetic sewage feed was diluted to 250 ml with water. 250 ml of sludge were added and the mixture was aerated continuously for 3 hours by bubbling air into it.

Control C6: at time "210 minutes", in a 500-ml scaled cylinder, 16 ml of the synthetic sewage feed was diluted to 250 ml with water. 250 ml of sludge were added and the mixture was aerated continuously for 3 hours by bubbling air into it.

Measurements of the oxygen consumption: at time "3 hours", the pH of the first cylinder C1 was measured. The content was used to fill up the BOD flask which was closed with the O2-electrode and the measurement of the oxygen consumption was started for about 10 minutes. The same procedure was repeated every 15 minutes for each cylinder whit bubbling air in the order they had been prepared, so that the contact time is three hours for each of them.

The whole experiment was carried out at 20 ± 2°C (temperature controlled water bath).
Test organisms (species):
other: synthetic sewage feed
Details on inoculum:
Synthetic sewage feed :
A synthetic sewage feed was made by dissolving the following amounts of substances in 1 litre of tap water:

- 16.0 g peptone (from casein trypsin-digested)
- 11.0 g yeast extract powder
- 3.0 g urea
- 0.7 g NaCl
- 0.4 g CaCl2 · 2 H2O
- 0.3 g MgSO4 · 7 H2O
- 2.8 g K2HPO4


Activated sludge :
Activated sludge was obtained from a water treatment plant treating predominantly domestic sewage (Bois-de Bay, 1242 Satigny, Switzerland). The sludge was kept aerobic until being used on the next day. The pH was 7.5.

The suspended solids content was determined by drying 50 ml of the sludge and weighing: dry weight of suspended solids: 5.00 g/l.

By adding 1670 ml of water to 2500 ml sludge, a concentration of suspended solids of 3.0 g/l was obtained. With the final dilution during the test, a concentration (i.e. in the range 0.8 to 1.6 g/l as recommended in ISO 8192-1986) was obtained.

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Results with reference substance (positive control):
3,5-dichlorophenol was used as a reference substance to check the activity of the sludge: its EC50 ( 3 hours) should be withi the range of 2 to 25 mg/L.

Observed :
2.0 mg/L 0.9 to 8.7 % inhibition of respiration
25.0 mg/L 72.6 to 79.1% inhibition of respiration

Validity criteria fulfilled:
yes
Conclusions:
An activated sludge respiration inhibition test was performed with GR-88-0778. In a limit test a test concentration of 100 mg/l was tested.

The result can be expressed as follows: The EC50 (3 hours) of GR-88-0778 is > 100 mg/L.

Description of key information

An activated sludge respiration inhibition test was performed with GR-88-0778. In a limit test a test concentration of 100 mg/l was tested. The result can be expressed as follows:


 

The EC50 (3 hours) of GR-88-0778 is > 100 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:
10 mg/L

Additional information