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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From June 19th, 2018 to November 1st, 2018
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))
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
The test concentrations were prepared without using a stock solution. The replicates of the test concentrations were prepared by direct addition of the test item to the mixture of deionised water and synthetic sewage feed just before adding the microbial inoculum.

Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Name and location of sewage treatment plant where inoculum was collected: sewage treatment works in Flörsheim/Main (Germany) treating predominantly domestic sewage.
- Washing medium for activated sludge: reconstituted water (OECD 1992).
- Washing procedure: the activated sludge was centrifuged for 2 minutes at 1000 rpm (corresponding to 196 g according to the centrifuge specifications); the clear supernatant was decanted and the activated sludge pellet was re-suspended in washing medium; the procedure was repeated two more times.
- Frequency of washing: three times.
- Storage time before test start: 2 days.
- Storage temperature: 20±2°C.
- Mixed liquor suspended solids level in the microbial inoculum : 3.86 g/L.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
19.2–19.7°C (definitive test)
20.0–21.0°C (supplemental test)
pH:
7.1–7.2 (definitive test)
7.1–7.4 (supplemental test)
Nominal and measured concentrations:
Preliminary range-finding test (nominal concentration): 10, 100 and 1000 mg test item/L including a nitrification inhibition control.
Definitive test (nominal concentration): 10.0, 31.6, 100, 316 and 1000 mg test item/L, equivalent to 2.5, 7.9, 25.1, 79.3 and 251 mg active ingredients/L.
Supplemental test (nominal concentration): 1000, 2000 and 4000 mg test item/L, equivalent to 251, 501 and 1004 mg active ingredients/L.
Details on test conditions:
TEST SYSTEM
- Test vessel: 1000 mL glass beaker .
- Type: closed test vessels were covered with watch-glasses.
- Fill volume: 500 mL.
- Aeration: air flow of 0.5-1 L/min.
- No. of vessels per concentration (replicates) for the definitive and supplemental tests: 3.
- No. of vessels per control (replicates): 6.
- Mixed liquor suspended solids level in the final test mixtures: 1.54 g/L.
- Volume of microbial inoculum per test mixture: 200 mL.
- Volume of synthetic sewage feed per test mixture: 16 mL.

TEST MEDIUM / WATER PARAMETERS
- Dilution water: deionised water.

EFFECT PARAMETERS MEASURED:
- pH: before starting the exposure (i.e. adding the microbial inoculum), the pH was measured in the test solutions of the definitive test and the supplemental test.
- Respiration measurement: the oxygen content of the test solutions was determined using a six-channel oxygen meter. The oxygen measurements were made on the content of each test vessel at the end of the contact period at 2-second intervals over a period of 5–10 minutes or until the oxygen concentration fell below 2 mg/L.

TEST CONCENTRATIONS
- Range finding study : yes.
- Test concentrations: 10, 100 and 1000 mg test item/L including a nitrification inhibition control.
- Results used to determine the conditions for the definitive study: yes.
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 4 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 1 004 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
inhibition of total respiration
Details on results:
RANGE FINDING TEST
For total respiration, the range-finding test showed no inhibition of control respiration at up to 1000 mg test item/L (equivalent to 251 mg active ingredient/L). An inhibition of nitrification compared to the control respiration was not found.

DEFINITIVE TEST
An inhibition of respiration rate was not observed up to and including the highest tested concentration level, 1000 mg test item/L (equivalent to 251 mg active ingredients/L).

SUPPLEMENTAL TEST
An inhibition of respiration rate was not observed up to and including the highest tested concentration level, 4000 mg test item/L (equivalent to 1004 mg active ingredients/L).

Biological results

Table 1: Decrease of total respiration rate (inhibition in % of control) as caused by the test item after 3 h.

Treatment

[mg test item/L]

Mean respiration rate

[mg O2/(l*h)]

Standard deviation

Inhibition of Respiration rate [% of control value]

Control

68.1

4.96

0

10

60.7

1.27

10.9

31.6

69.1

1.82

-1.4

100

67.8

3.24

0.5

316

71.2

3.28

-4.5

1000

77.8

3.97

-14.3

 

 Table 2: Decrease of total respiration rate (inhibition in % of control) as caused by the test item after 3 h.

Treatment

[mg test item/L]

Mean respiration rate

[mg O2/(l*h)]

Standard deviation

Inhibition of Respiration rate [% of control value]

Control

44.0

3.24

0

1000

51.7

5.63

-17.3

2000

56.4

4.20

-28.2

4000

47.9

7.48

-8.8

Validity criteria fulfilled:
yes
Remarks:
All validity criteria were fulfilled (coefficient of variation of O2 consumption rate in control replicates: ≤30%; O2 consumption rate in the controls: ≥20 mg O2/(g dw × h); 3h-EC50 of 3,5-dichlorophenol in the accepted range of 2–25 mg/L).
Conclusions:
The definitive test showed no concentration-response relationship with no inhibition of total respiration rate compared to the control respiration up to and including 1000 mg test item/L (equivalent to 251 mg active ingredients/L). Since there was no inhibition of total respiration up to and including 4000 mg test item/L (equivalent to 1004 mg active ingredient/L) in a supplemental test, ECx values could not be determined. The results of this study show that the EC20, EC50, EC80 are > 4000 mg test item/L (equivalent to 1004 mg active ingredient/L).
Executive summary:

The effect of the REACH substance "Stearic acid, reaction product with 2-aminoethanol, maleic anhydride and sodium sulfite" (UVCB) on the respiration rate of activated sludge bacteria was investigated in a GLP-compliant study performed according to OECD Guideline 209. The respiration rate of the test system was measured after a 3-hour contact period with the test item. The effect of the test item on the respiration of nitrifying bacteria was investigated in a preliminary non-GLP range-finding test.

The preliminary non-GLP range-finding test including a nitrification inhibition control showed no inhibition of total respiration at up to 1000 mg test item/L (equivalent to 251 mg active ingredients/L); an inhibition of nitrification-related respiration was not observed in the range-finding test. The definitive test showed no concentration-response relationship with no inhibition of total respiration rate compared to the control respiration at up to 1000 mg test item/L. A supplemental test was performed at 1000, 2000 and 4000 mg test item/L to investigate potential effects on the respiration rate at 2000 and 4000 mg test item/L, and to confirm the absence of effects on the respiration rate at 1000 mg test item/L as seen in the definitive test. This supplemental test showed no inhibition of total respiration. The results of this study show that the EC20, EC50, EC80 are > 4000 mg test item/L (equivalent to 1004 mg active ingredients/L).

Description of key information

The effect of the REACH substance "Stearic acid, reaction product with 2-aminoethanol, maleic anhydride and sodium sulfite" (UVCB) on the respiration rate of activated sludge bacteria was investigated in a GLP-compliant study performed according to OECD Guideline 209. The respiration rate of the test system was measured after a 3-hour contact period with the test item. The effect of the test item on the respiration of nitrifying bacteria was investigated in a preliminary non-GLP range-finding test.

The preliminary non-GLP range-finding test including a nitrification inhibition control showed no inhibition of total respiration at up to 1000 mg test item/L (equivalent to 251 mg active ingredients/L); an inhibition of nitrification-related respiration was not observed in the range-finding test. The definitive test showed no concentration-response relationship with no inhibition of total respiration rate compared to the control respiration at up to 1000 mg test item/L. A supplemental test was performed at 1000, 2000 and 4000 mg test item/L to investigate potential effects on the respiration rate at 2000 and 4000 mg test item/L, and to confirm the absence of effects on the respiration rate at 1000 mg test item/L as seen in the definitive test. This supplemental test showed no inhibition of total respiration. The results of this study show that the EC20, EC50, EC80 are > 4000 mg test item/L (equivalent to 1004 mg active ingredients/L).

Key value for chemical safety assessment

EC50 for microorganisms:
1 004 mg/L

Additional information

One reliable experimental study is available and is selected as key study (ECT, 2018). It was performed according to OECD Guideline 209 and under GLP principles.