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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 Nov - 01 Dec 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
Version / remarks:
2010
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A stock solution of 10 g/L was prepared by adding 5.0025 g test item to 500 mL of Milli-RO water (tap water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA). Magnetic stirring for 10 - 17 min was applied to accelerate dissolution and to ensure homogeneity. Volumes of the clear slightly pink stock solution (pH 1.8) corresponding to the test concentration were then added to the test media (16 mL synthetic medium and made up to 250 mL with Milli- RO). Thereafter, 250 mL of activated sludge was added resulting in the required concentrations. Optimal contact between the test item and test organisms was ensured by applying continuous aeration and stirring.
- Controls: Yes
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Name and location of sewage treatment plant where inoculum was collected: Municipal sewage treatment plant: 'Waterschap Aa en Maas', 's -Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.
- Preparation of inoculum for exposure: The sludge was coarsely sieved (1 mm) and allowed to settle. The supernatant was removed and ISO-medium was added. The batch of sludge was used one day after collection; therefore 50 mL of synthetic medium (=sewage feed) was added per litre of activated sludge at the end of the collection day. The sludge was kept aerated at test temperature until use.
- Pretreatment: A small amount of the sludge was weighed and dried overnight at ca. 105 °C to determine the amount of suspended solids (3.0 g/L of sludge, as used for the test). The pH was 7.7 on the day of testing.
- Initial biomass concentration: 3.0 g/L of sludge, as used for the test
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
19 - 21 °C
pH:
7.3 - 7.9 (Control)
4.4 - 7.9 (Test item concentrations)
Dissolved oxygen:
see Table 1 in section "Any other information on results incl tables"
Nominal and measured concentrations:
Control, 3.2, 10, 32, 100 and 320 mg/L (nominal)
Details on test conditions:
TEST SYSTEM
Test vessel:
- Type: open
- Material, size, headspace, fill volume: glass vessel with a volume of 500 mL
- Aeration: The aeration was adjusted in such a way that the dissolved oxygen concentration at the start was above 60-70% saturation (60% of air saturation is > 5 mg/L at 20°C) and to maintain the sludge flocs in suspension; Clean, oil-free air. Determination of oxygen was performed with multiple oxygen sensors connected to a BlueBox (GO-Systemelektronik GmbH, Germany), a multichannel measuring and controlling system.
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 6
- Sludge concentration (weight of dry solids per volume): 3.0 g/L (corresponding to 250 mL)
- Nutrients provided for bacteria: synthetic medium (sewage feed) according to the guideline (ISO medium)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Adjusted ISO-medium, formulated using RO water (tap water purified by reverse osmosis; GEON Waterbehandeling, Berkel-Enschot, The Netherlands)

EFFECT PARAMETERS MEASURED: After the 3 h contact time the oxygen consumption was recorded for a period of approximately 10 min.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
Range finding study
- Test concentrations: Control, 10, 100 and 1000 mg/L
- Results used to determine the conditions for the definitive study:
The combined limit/range-finding showed 11%, 58% and 85% inhibition of the respiration rate at a concentration of 10, 100 and 1000 mg/L, respectively. Therefore, the expected EC50 was at a concentration of approximately 100 mg/L. There was no significant oxygen uptake from abiotic processes. All test conditions and acceptability criteria prescribed by the study plan were met.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
3.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
7.7 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks on result:
other: 95% confidence interval: 5.5 - 10 mg/L
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
92 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks on result:
other: 95% confidence interval: 80 - 106 mg/L
Duration:
3 h
Dose descriptor:
other: EC20
Effect conc.:
18 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks on result:
other: 95% confidence interval: 14 - 22 mg/L
Details on results:
- Blank controls oxygen uptake rate: 24 mg oxygen per one gram of activated sludge in the final test
- Coefficient of variation of oxygen uptake rate in control replicates: 10%
Results with reference substance (positive control):
The EC50 of 3,5-dichlorophenol was with 5.0 mg/L in the accepted range of 2 to 25 mg/L for total respiration
Reported statistics and error estimates:
Calculations were performed with ToxRat Professional v. 3.2.1 (ToxRat Solutions® GmbH, Germany). For the test item, calculation of the ECx value was based on probit analysis using linear maximum likelihood regression, with the percentages of respiration inhibition versus the logarithms of the corresponding concentrations of the test item. No statistically significant inhibition of the respiration rate of the sludge was recorded at 3.2 mg test item/L. At higher concentrations the inhibitory effect of the test item on aerobic waste water (activated sludge) bacteria increased with increasing concentration, ranging from 19% inhibition at 10 mg/L to 79% at 320 mg/L.

Table 1: Main Test – Overview of the Results

Treatment

Concentration

(mg/L)

Mean respiration rate

% Inhibition of the respiration rate (mean value)

(mg O2/L h)

(mg O2/g h)¹

Control

 

36.16

24.10

 -

T1

3.2

34.87

23.25

3.55

T2

10

29.22

19.48*

19.20

T3

32

27.68

18.45*

23.46

T4

100

18.37

12.25*

49.20

T5

320

7.72

5.15*

78.65

¹) The amount of suspended solids in the final test mixture was 1.5 g/L.

* Statistically significantly different compared to control.

Table 2: Results of the main Test – Respiration Rate/Inhibition, pH Values

Replicate

Concentration

(mg/L)

pH

Respiration rate

% Inhibition

respiration rate

(mean value)

Start

End

(mg O2/L.h)

(mg O2/g.h)¹

 

 

 

 

 

 

 

C 1

0

7.5

7.9

33.24

22.16

C 2

0

7.3

7.7

38.31

25.54

C 3

0

7.3

7.7

40.36

26.91

C 4

0

7.3

7.7

32.42

21.61

C 5

0

7.3

7.7

39.35

26.23

C 6

0

7.3

7.7

33.26

22.17

C Mean

 

 

 

36.16

24.10 (RC)

 

SD

 

 

 

3.56

2.37

 

CV (%)

 

 

 

10

10

 

 

 

 

 

 

 

 

R 1

1.0

7.4

8.0

34.79

23.19

3.78

R 2

3.2

7.5

8.2

18.75

12.50

48.14

R 3

10

7.5

8.1

14.19

9.46

60.75

R 4

32

7.5

8.2

6.07

4.05

83.21

 

 

 

 

 

 

 

T 1a

3.2

7.4

7.8

38.99

25.99

-7.84

T 1b

3.2

7.4

7.9

37.54

25.03

-3.83

T 1c

3.2

7.4

7.9

31.30

20.87

13.43

T 1d

3.2

7.4

7.9

28.58

19.05

20.96

T 1e

3.2

7.4

7.7

37.96

25.31

-4.99

T 1 Mean

 

 

 

34.87

23.25 (RT)

3.55 (IT)

 

 

 

 

 

 

 

T 2a

10

7.3

7.9

29.93

19.95

17.22

T 2b

10

7.3

7.9

31.11

20.74

13.96

T 2c

10

7.3

7.8

27.67

18.45

23.47

T 2d

10

7.3

7.8

29.03

19.35

19.71

T 2e

10

7.3

7.9

28.34

18.89

21.62

T 2 Mean

 

 

 

29.22

19.48 (RT)*

19.20 (IT)

 

 

 

 

 

 

 

T 3a

32

7.1

7.8

30.19

20.13

16.50

T 3b

32

7.1

7.9

26.94

17.96

25.49

T 3c

32

7.1

7.8

24.62

16.41

31.91

T 3d

32

7.1

7.7

26.87

17.91

25.68

T 3e

32

7.1

7.7

29.76

19.84

17.69

T 3 Mean

 

 

 

27.68

18.45 (RT)*

23.46 (IT)

 

 

 

 

 

 

 

T 4a

100

6.4

7.8

15.77

10.51

56.38

T 4b

100

6.4

7.7

20.86

13.91

42.31

T 4c

100

6.4

7.7

18.37

12.25

49.19

T 4d

100

6.4

7.7

18.07

12.05

50.02

T 4e

100

6.4

7.7

18.77

12.51

48.09

T 4 Mean

 

 

 

18.37

12.25 (RT)*

49.20 (IT)

 

 

 

 

 

 

 

T 5a

320

4.4

7.6

7.48

4.99

79.31

T 5b

320

4.4

7.6

7.85

5.23

78.29

T 5c

320

4.4

7.6

7.33

4.89

79.73

T 5d

320

4.4

7.5

7.56

5.04

79.09

T 5e

320

4.4

7.6

8.38

5.59

76.82

T 5 Mean

 

 

 

7.72

5.15 (RT)*

78.65 (IT)

C:       Control

SD:    Standard deviation

CV:    Coefficient of variation

R:       Reference item, 3,5-dichlorophenol

RTB:  Total respiration in the control

T:       Test item

¹:        The amount of suspended solids in the final test mixture was 1.5 g/L

RT:     Total respiration with test item

IT:      % inhibition of total respiration relative to RC

*:        Statistically significantly different compared to control

Table 3: Validity criteria 

Criterion from the guideline

Outcome

Validity criterion fulfilled

The blank controls (without the test substance or reference substance) oxygen uptake rate should not be less than 20 mg oxygen per one gram of activated sludge (dry weight of suspended solids) in an hour.

24 mg oxygen per one gram of activated sludge

Yes

The coefficient of variation of oxygen uptake rate in control replicates should not be more than 30% at the end of definitive test.

10%

Yes

EC50 of 3,5-dichlorophenol shoukld be within the range of 2 – 25 mg/L for total respiration

5.0 mg/L

Yes

 

Validity criteria fulfilled:
yes
Remarks:
For further details please see table 3 in section: "Any other information on results incl tables".
Conclusions:
The test item was not toxic to waste water (activated sludge) bacteria at a concentration of 3.2 mg/L or below. The EC10 was determined to be at 7.7 mg/L.

Description of key information

EC10 (3 h) = 7.7 mg/L (95% confidence interval: 5.5 -10 mg/L, activated sludge, OECD 209)

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
7.7 mg/L

Additional information

One experimental study was conducted in accordance with OECD Guideline 209 and EU method C.11 under GLP conditions (Buitenweg, 2018b). The activated sludge was exposed to the test item concentrations of 0, 3.2, 10, 32, 100 and 320 mg/L. Five replicates per concentration and six replicates for an untreated control group were tested. No statistically significant inhibition of the respiration rate of the sludge was recorded at 3.2 mg of the test item/L. At higher concentrations the inhibitory effect of the test item on aerobic waste water (activated sludge) bacteria increased with increasing concentration, ranging from 19% inhibition at 10 mg/L to 79% at 320 mg/L. The EC10 and EC50 (3 h) values are 7.7 and 92 mg/L, respectively.