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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
September - December, 2014
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
ISO 8192 (Water quality - Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation)
Version / remarks:
1986
Qualifier:
according to guideline
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Version / remarks:
2008
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Version / remarks:
2010
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
GR-50-1408 is the Givaudan identification code which was employed for ROSYFOLIA during the early, developmental and testing period.
Vehicle:
no
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
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 8.0.

The suspended solids content was determined by drying 50 ml of the sludge and weighing: dry weight of suspended solids: 8.90 g/l.

By adding 2950 ml of water to 1500 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.

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
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Test temperature:
The whole experiment was carried out at 20 ± 2°C (temp controlled water bath)
pH:
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 8.0.
Nominal and measured concentrations:
The test item was tested as a limit test at the nominal test concentration of 100 mg/L.
Details on test conditions:
The respiration rate of an activated sludge fed with a standard amount of synthetic sewage feed is measured after a contact time of 3 hours.
The respiration rate of the same activated sludge in the presence of several concentrations of the test substance under otherwise identical conditions is also measured.
The inhibitory effect of the test substance at a particular concentration is expressed as a % of the mean respiration rates of 6 controls. An EC50 value if calculated from determinations performed on a range of test substance conditions.
 
Note: the highest conc tested is 100mg/L
 
3,5-dichlorophenol was used as a reference substance to check the activity of the sludge: its EC50 (3hours) should be within the range 2-25mg/L.
 
A synthetic sewage feed was made by dissolving the following amounts of substances in 1 litre of tap water:
16.0g peptone (from casein trypish-digested)
11.0g yeast exract powder
3.0g urea
0.7g NaCl
0.4g CaCl2 – 2 H2O
0.3g MgSO4 – 7H2O
2.8g K2HPO4
 
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 8.0.
The suspended solids content was determined by drying 50ml of the sludge and weighing: dry weight of suspended solids: 8.90g/l.
By adding 2950ml of water to 1500ml sludge, a concentration of suspended solids of 3.0g/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.
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 on result:
other: The average % inhibition at the limit test concentration of 100 mg/L was 34.2% (29.5%, 36.6%, 36.6%)
Details on results:

Conditions for the validity of the test:
- The variation coefficient of the controls was 15.0%. This is within the validity range (30%).
- the EC50 (3 hours) of 3,5-dichlorophenol is between 2 and 25 mg/L.

The tested concentration of the test item (100 mg/l) produced inhibitions below 50% (range 29.5 to 36.6%)


Therefore:
The EC50 (3 hours) of GR-50-1408 is > 100 mg/l
Results with reference substance (positive control):
The EC50 (3 hours) of 3,5-dichlorophenol is between 2 and 25 mg/l :
- Average Inhibition % at 2 mg/L = 20.1%
- Average Inhibition at 25 mg/L = 56.2%

The results of the test are tabulated below.

Conditions for the validity of the test:

The variation coefficient of the controls was 15.0%. This is within the validity range (30%) and the EC50 (3hours) of 3,5-dichlorophenol is between 2 and 25 mg/L.

 

vessel

pH

O2 (mg/l)

Time (min)

rate

%

inhibition

Conc. Final (mg/l)

Log (conc)

C1

8.5

1.54

5.0

0.31

 -

 -

 -

C2

8.4

1.54

5.0

0.31

 -

 -

 -

C3

8.4

1.55

5.0

0.31

 -

 -

 -

S1

8.1

1.25

5.0

0.25

29.5

100.0

2.00

S2

8.2

1.13

5.0

0.23

36.6

100.0

2.00

S3

8.3

1.13

5.0

0.23

36.6

100.0

2.00

R1

8.4

1.38

5.0

0.28

22.5

2.0

0.30

R2

8.3

1.38

5.0

0.28

22.5

2.0

0.30

R3

8.4

1.50

5.0

0.30

15.4

2.0

0.30

R4

8.4

0.88

5.0

0.18

50.7

25.0

1.40

R5

8.4

0.75

5.0

0.15

57.7

25.0

1.40

R6

8.5

0.71

5.0

0.14

60.2

25.0

1.40

C4

8.4

1.88

5.0

0.38

 -

 -

 -

C5

8.4

2.13

5.0

0.43

 -

 -

 -

C6

8.5

2.00

5.0

0.40

 -

 -

 -

Validity criteria fulfilled:
yes
Conclusions:
The tested concentration (100 mg/L) produced inibitions below 50% (range 29.5 to 36.6%).
Therefore EC50(3h) of GR-50-1408 > 100 mg/L

Description of key information

An activated sludge respiration inhibition test was performed with GR-50-1408 in line with OECD Guideline for the Testing of Chemicals No. 209. In a limit test a test concentration of 100mg/L was tested. The results can be expressed as follows:

The EC50(3h) of GR-50-1408 > 100 mg/L

Key value for chemical safety assessment

EC50 for microorganisms:
100 mg/L

Additional information