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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Version / remarks:
2010
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
Three nominal concentrations were tested: 10, 100 and 1000 mg/L; single test vessels were tested for the two lower concentrations, and three replicates were tested for the concentration of 1000 mg/L. A solubility pre-experiment showed that the test substance was soluble at a concentration of at least 5 g/L in deionised water, before and after adjustment of the pH from 2.2 to 7.9. A stock solution with a concentration of 5 g/L was prepared and the pH was adjusted from 2.2 to 7.3 with a sodium hydroxide solution. The test solutions were then prepared from the stock solution by dilution with deionised water.
The positive reference substance 3,5-dichlorophenol was tested at concentrations of 3.2, 10 and 32 mg/L (one vessel per concentration).
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Name and location of sewage treatment plant where inoculum was collected:
ARA Birs, Birsfelden, Switzerland
- Method of cultivation: wet sludge was suspended in chlorine free tap water to achieve a concentration of 3 g dry material per litre. During the holding period of one day before use, the sludge was fed wonce with 50 mL synthetic sewage per litre
- Preparation of inoculum for exposure: before use on the next day, the pH of the activated sludge inoculum was adjusted from pH 6.9 to pH 7.5 with a 1M NaOH solution
- Initial biomass concentration: 3 g dry material per litre
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
19 to 21 °C
pH:
Control vessels: 7.4 to 7.5 at start and 7.9 to 8.0 at end
Test substance vessels: 7.4 to 7.5 at start and 7.9 to 8.1 at end
Reference substance vessels: 7.2 to 7.5 at start and 7.9 to 8..1 at end
Dissolved oxygen:
Control vessels: 8.5 to 8.6 mg/L at start and 7.8 to 8.0 mg/L at end
Test substance vessels: 8.2 to 8.5 mg/L at start and 7.8 to 8.2 mg/L at end
Reference substance vessels: 8.4 to 8.6 mg/L at start and 8.4 to 8.9 mg/L at end
Nominal and measured concentrations:
Test substance: 10, 100, 1000 mg/L
Positive control: 3.2, 10, 32 mg/L
Details on test conditions:
Synthetic sewage feed was prepared to contain 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 7H20 and 2.8 g K2HPO4 per litre. The pH of the solution was 7.2.
Control vessels were prepared by using 16 mL synthetic sewage feed and making up to 250 mL with ionised water. Then 250 mL activated sewage sludge concentration with a nominal concentration of 3 g dry material per litre were added.
The test vessels were prepared by diluting proper volumes of the stock solution with deionised water, then adding 16 mL synthetic sewage feed and 250 mL of the activated sewage sludge inoculum.
The positive control vessels were prepared by mixing appropriate aliquots of a 3,5-DCP stock solution at a concentration of 500 mg/L with deionised water, synthetic sewage feed and inoculum in the respective test vessels to obtain the desired test concentrations.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Key result
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
Details on results:
The specific respiration rates for the two blank control replicates prepared at the beginning of the exposure period were 36 and 34 mg O2 per dry weight of activated sludge per hour and for two blank controls prepared at the end of the test were 34 and 39 mg O2/gh. All four values are higher than the 20 mg O2/gh stipulated in the guidelines.
The resulting coefficient of variation of oxygen uptake rate in the control replicates has a value of 6% and is therefore, as stipulated in the guidelines, not more than 30%.
Results with reference substance (positive control):
The 3-hour EC50 of the reference item 3,5-dichlorophenol was calculated to be 6.6 mg/L with 95% confidence limits from 6.2 to 6.9 mg/L. The 3-hour EC50 is within the guidelines-recommended range of 2 to 25 mg/L, confirming the suitability of the activated sludge used.

Table 1: Results of the study

Vessel no.

Nominal test concentration (mg/L)

Oxygen consumption rate

Inhibition %

pH values

Oxygen concentration (mg O2/L)

R in mg O2/L.h

Rs in mg O2/g.h

start

end

start

end

Control

1

0

54.74

36.49

 

7.5

7.9

8.5

8.0

2

0

50.86

33.91

 

7.5

7.9

8.5

8.2

11

0

50.77

33.85

 

7.4

8.0

8.6

8.1

12

0

57.90

38.60

 

7.4

8.0

8.5

7.8

Mean

Standard deviation

Coefficient of variation

53.57

3.43

6.40%

35.71

2.29

6.40%

 

 

 

 

 

Test substance

6

10

53.45

35.63

0.2

7.5

8.0

8.4

7.9

7

100

50.56

33.71

5.6

7.5

7.9

8.2

8.0

8

1000

49.04

32.69

8.5

7.5

8.1

8.3

7.8

9

1000

49.75

33.17

7.1

7.4

8.1

8.5

8.2

10

1000

54.00

36.00

-0.8

7.4

7.9

8.5

7.8

Positive reference substance

3

3.2

38.10

25.40

28.9

7.2

8.1

8.6

8.4

4

10

19.91

13.27

62.8

7.5

8.0

8.5

8.5

5

32

6.00

4.00

88.8

7.4

7.9

8.4

8.9

Validity criteria fulfilled:
yes
Conclusions:
The 3-hour NOEC for the inhibitory effect on the respiration of activated sludge inoculum in the study was 1000 mg/L.
Executive summary:

The inhibitory effect of the substance on the respiration rate of aerobic wastewater micro-organisms of activated sludge was investigated under GLP in accordance with OECD TG 209 in a 3-hour respiration inhibition test. A range finding test using concentrations of 10, 100 and 1000 mg test substance/L was performed. Single test vessels were used for the two lower concentrations, and three test vessel replicates were used at 1000 mg/L. The positive reference substance 3,5-dichlorophenol was also tested under the same conditions, at concentrations of 3.2, 10 and 32 mg/L. Four blank controls, containing an equal volume of activated sludge and synthetic sewage medium but no test or reference substances, were included in the test. The vessels were prepared in this order: two blank controls, positive reference substance vessels (with ascending concentrations, one vessel per concentration), test substance vessels (with ascending concentrations), and finally two blank controls. The test temperature was between 19 and 21 °C, the pH value in all test vessels was between 7.2 and 7.5 at the start and between 7.9 and 8.1 at the end, and the dissolved oxygen concentration ranged from 8.2 to 8.6 mg/L at the start and 7.8 to 8.9 mg/L at the end. All validity criteria of the test were fulfilled: the specific respiration rate of the blank controls was between 34 and 39 mg oxygen per gram dry weight of sludge per hour (thus >20 mg O2/g.h), the coefficient of variation of oxygen uptake rate in the control replicates was 6% (thus <30%), and the 3-hour EC50 of the positive reference substance was 6.6 mg/L (thus in the acceptable range from 2 to 25 mg/L). The test substance had no statistically significant inhibitory effect on the respiration rate of the activated sludge. The 3-hour NOEC in this study was 1000 mg/L.

Description of key information

3-hour NOEC = 1000 mg/L (GLP, OECD TG 209)

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
1 000 mg/L

Additional information