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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29. May- 30 May 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
Version / remarks:
2010
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Version / remarks:
No. 2016/266 amending Regulation (EC) No. 440/2008 adopted 07 Dec 2015
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht Rheinland-Pfalz, Mainz, Germany
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test item was added to the test vessels directly.
- Eluate: No
- Differential loading: Yes
- Controls: Yes (only medium and activated sludge)
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Name and location of sewage treatment plant where inoculum was collected: Activation basin of the ESN (Stadtentsorgung Neustadt) sewage treatment plant in D-67435 NW-Lachen-Speyerdorf, Germany
- Preparation of inoculum for exposure: On the day before the experiment, the inoculum was taken from its source, washed, aerated and the dry matter was determined. Volume was adapted to the desired content of dry matter. The nutrient solution was thawed and the sludge was fed with 50 mL nutrient solution/L sludge.
- Pretreatment: Sludge was filtrated, washed with tap water 3 times and re-suspended in tap water. The activated sludge was aerated until usage in the test and fed daily with 50 mL synthetic sewage feed /L
- Initial biomass concentration: 1.58 g suspended solids/L dry matter in the test
- Dry matter of sludge concentration: 3.16 g suspended solids/L
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
20.4 – 21.2 °C
pH:
7.8 - 7.9 (Control)
7.8 (Test item concentrations)
Nominal and measured concentrations:
Control, 1, 10, 100 and 1000 mg/L
Details on test conditions:
TEST SYSTEM
Test vessel:
- Type: open
- Material, size, headspace, fill volume: Glass beakers (800 – 1000 mL) were used as test vessels and narrow-neck glass bottles with flat bottoms (250 mL) were used as measuring flasks
- Aeration: Activated sludge was aerated until usage in the test, purified air, using Pasteur pipettes
- No. of vessels per concentration (replicates): 5 (for highest test item concentration1000 mg/L) and 1 for the lower test item concentrations (1, 10 and 100 mg/L)
- No. of vessels per control (replicates): 6
- Sludge concentration (weight of dry solids per volume): 1.58 g suspended solids/L
- Nutrients provided for bacteria: Nutrient solution, 16 mL/vessel, synthetic sewage according to guideline OECD 209

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap water (pH 8.16; Conductivity at 25 °C: 228 µS/cm; Hardness: 0.83 mmol/L)
- Particulate matter: TOC < 0.50 mg/L

EFFECT PARAMETERS MEASURED: O2 consumption measured over a period of max 5 min and the pH are measured at the end of the test

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10 range-finding test in a limit test design
Range finding study : The test was a combination of range-finding test and limit test
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol
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
Duration:
3 h
Dose descriptor:
EC10
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:
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:
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No
- Blank controls oxygen uptake rate: 25.12 mg oxygen per gramm sludge in 1 h, see also table in "Any other information on results incl table"
- Coefficient of variation of oxygen uptake rate in control replicates: 2.5% , see also table in "Any other information on results incl table"
Results with reference substance (positive control):
EC50 7.4 mg/L (95% confidential limits: 5.0 - 9.6 mg/L). The value lies within the recommended range of 2 – 25 mg/L.
Reported statistics and error estimates:
Following the t-value WEIR-Test the difference between treatment 1000 mg/L and the blank control can be considered as not significant as the calculated t-value lay below the tabulated t-value. Therefore, the concentration 1000 mg/L is stated as NOEC.

Table 1: O2-Consumption, Inhibition

Vessel No.

Content

Concentration in mg/L (nominal)

O2 consumption in mg/(L*min)

O2 consumption in mg/(L*h)

Inhibition in %

pH

1

Blank Control

0

0.6703

40.215

0

7.9

2

Blank Control

0

0.6425

38.552

0

7.9

3

PositiveControl

5

0.4138

24.828

37.5

8.0

4

Positive Control

10

0.2678

16.066

59.5

8.0

5

Positive Control

20

0.1418

8.509

78.6

8.0

6

Positive Control

40

0.0830

4.980

87.5

8.0

7

Blank Control

0

0.6745

40.469

0

7.8

8

Blank Control

0

0.6399

38.395

0

7.8

9

Test Item

1000

0.6545

39.271

1.1

7.8

10

Test Item

1000

0.6521

39.129

1.4

7.8

11

Test Item

1000

0.6588

39.530

0.4

7.8

12

Test Item

1000

0.6605

39.631

0.2

7.8

13

Test Item

1000

0.6598

39.588

0.3

7.8

14

Test Item

100

0.6629

39.772

-0.2

7.8

15

Test Item

10

0.6698

40.189

-1.2

7.8

16

Test Item

1

0.6688

40.126

-1.1

7.8

17

Blank Control

0

0.6645

39.870

0

7.8

18

Blank Control

0

0.6777

40.660

0

7.8

Table 2: Validity criteria for OECD 209

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.

25.12 mg oxygen per gram sludge in 1 h

Yes

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

2.5%

Yes

 

Validity criteria fulfilled:
yes
Remarks:
See detailed information in section "Any other information on results incl tables"
Conclusions:
The test item was tested using 4 concentrations ranging from 1000 to 1 mg/L nominal concentration. For the treatment 1000 mg/L, 5 replicates were used; for the other treatments, 1 replicate each. Because no significant inhibition was observed, no additional experiment had to be performed.

Description of key information

NOEC (3 h) ≥ 1000 mg/L (OECD 209, activated sludge, nominal)

Key value for chemical safety assessment

Additional information

There is one GLP study available investigating the respiration inhibition of activated sludge microorganisms according to OECD guideline 209.

To investigate the inhibitory effects on total respiration, non-adapted activated sludge from a municipal sewage treatment plant was fed with synthetic sewage and inoculated with nominal concentrations of 1, 10, 100 and 1000 mg/L substance for 3 h under aerobic conditions. Blank controls (without test item) and sensitivity controls (with a reference item at three concentration levels) were carried out in parallel. After 3 h, the oxygen consumption rate was measured by inserting an oxygen electrode into a BOD bottle and recording oxygen concentration. No respiration inhibition of the activated sludge compared to the blank controls was determined up to 1000 mg/L. The endpoints EC50 and EC10 were reported as >1000 mg/L and the NOEC as ≥ 1000 mg/L.