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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-05-31 to 2017-06-01
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Version / remarks:
2010
Deviations:
no
Principles of method if other than guideline:
In the preliminary test, additional replicates were prepared to determine the inhibition of nitrification by measuring with addition of Allylthiourea.
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
no
Details on sampling:
not applicable (no analytics)
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: weighed out directly in the Erlenmeyer flasks

- Eluate: n.a.
- Differential loading: no
- Controls: reference item concentrations were diluted out of a stock solution.

- Chemical name of vehicle (organic solvent, emulsifier or dispersant): n.a.
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): n.a.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): not reported
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
Non-adapted activated sludge from the sewage plant at Hildesheim is weil suited as it receives predominantly municipal sewage and hardly any industrial chemical waste.

- Name and location of sewage treatment plant where inoculum was collected: municipal treatment plant of 31137 Hildesheim, Germany
- Pretreatment: The sludge was washed twice with chlorine free tap water and adjusted to a dry sludge concentration of 3.0 g/L ± 10 %. The sludge was used within 24 h after sampling.
- Initial biomass concentration: Dry sludge concentration 2.71 g/L (corresponding to 1.36 g/L suspended solids in the test vessels)
Test type:
static
Water media type:
other: 16 mL sythetic waste water (Synthetic waste water according to OECD-Guideline 209), filled up with demineralised water to 250ml
Limit test:
yes
Total exposure duration:
3 h
Hardness:
not reported
Test temperature:
actually measured 20.8 °C
pH:
activated sludge 7.45
sytnthetic waste water 7.34
test solution 7.66 (1000mg/l)
Dissolved oxygen:
6.85 - 7.56 (3 test item replicates, determined at start)
Salinity:
not reported
Conductivity:
not reported
Nominal and measured concentrations:
preliminary test: 10, 100, 1000 mg/L
main test: 1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 500 mL Erlenmeyer flasks, DIN 12380
- Material, size, headspace, fill volume: glas, 500ml, 250m
- Aeration: yes
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): 3
- No. of vessels per abiotic control (replicates): n.a.
- Sludge concentration (weight of dry solids per volume): Dry sludge concentration 2.71 g/L (corresponding to 1.36 g/L suspended solids in the test vessels)
- Nutrients provided for bacteria: Synthetic waste water according to OECD-Guideline 209
- Nitrification inhibitor used (delete if not applicable): N-allylthiourea (pre-liminary study)


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: demineralised water
- Particulate matter: n.a.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: not reported
- Light intensity: n.a.
- Details on termination of incubation: not reported

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
TEST CONCENTRATIONS
- Spacing factor for test concentrations: preliminary test: 10; main test: limit test
- Justification for using fewer concentrations than requested by guideline:
Based on the results of the preliminary test it could concluded that the heterotrophic respiration as well as the nitrification are not inhibited. Therefore, the definitive study was carried out with the concentration of 1000 mg/L in 3 replicates (limit test to determine the NOEC).

- Range finding study : yes
- Test concentrations: 10, 100, 1000 mg/l
- Results used to determine the conditions for the definitive study: no significant inhibition of respiration or nitrification in the definitive study was observed, even the inhibitions of the heterotrophic respiration or nitrification show no appreciable value.
Reference substance (positive control):
yes
Remarks:
Copper (II) sulphate pentahydrate
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:
- Adsorption (e.g. of test material to the walls of the test container): not measured / reported
- Blank controls oxygen uptake rate: 33.3 mg O2 / (Lxh)
- Coefficient of variation of oxygen uptake rate in control replicates: 1.92%
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: EC50=93.1mg/l for copper (II) sulphate pentahydrate
Reported statistics and error estimates:
Statistical Analysis (NOEC)

One Way Analysis of Variance
Normality Test (Shapiro-Wilk) Passed (P = 0.503)
Equal Variance Test: Passed (P = 0.462)

Group Name N Missing Mean Std Dev SEM
Control 6 0 33.300 0.701 0.286
1000 mg/L 3 0 34.833 0.802 0.463

Source of Variation DF SS MS F P
Between Groups 1 4.702 4.702 8.785 0.021
Residual 7 3.747 0.535
Total 8 8.449

The differences in the mean values among the treatment groups are greater than would be expected by chance; there is a statistically significant difference (P = 0.021).

Power of performed test with alpha = 0.050: 0.673


Multiple Comparisons versus Control Group (Dunnett's Method) :
Comparisons for factor:
Comparison Diff of Means q' P P<0,050
Control vs. 1000 mg/L 1.533 2.964 -- Yes

Preliminary Test (non GLP)

 

Inhibitions in the Preliminary Test

Nominal Test Item Concentration
[mg/L]

pH

(test item in medium)

Inhibition


[%]

1stpreliminary test - without pH adjustment, direct aeration

10

-

-4

-

-2

100

-

-1

-

0

1000

7.66

-1

-

-4

Inhibitions in the Preliminary Test (with / without ATU)

Nominal Test Item Concentration
[mg/L]

Inhibition of total respiration
[%]

Inhibition of heterotrophic respiration
[%]

Inhibition of nitrification
[%]

1000

-1

-19

10

-4

-16

3

 

Oxygen Uptake Rates, Specific Respiration Rates and Inhibition of the Respiration

From the oxygen depletion the oxygen uptake rates were calculated in mg O2/L×h, additionally the specific respiration rates based on the dry sludge concentration were calculated in mg O2/g×h. The inhibition of the oxygen uptake rates of the test item concentrations in % was calculated in comparison to the oxygen uptake rates of the control.

 

Oxygen Uptake Rates and Specific Respiration Rates of the Control Replicates

 

Repl.

Oxygen
Concentration*

Oxygen Uptake Rate R

Specific Respiration Rate Rs

 

 

[mg O2/L]

[mg O2/(L×h)]

[mg O2/g×h]

Control

1

7.88

32.8

24.2

2

7.93

33.4

24.6

3

7.24

32.6

24.0

4

8.06

34.4

25.4

5

8.67

33.8

24.9

6

8.41

32.8

24.2

 

Mean value

33.3

24.6

Standard deviation

0.640

 

CV [%]

1.92

*) determined at start of the measurement

 Repl. = Replicate

CV = Coefficient of variation

Oxygen Uptake Rates and Inhibition of the Respiration of the Test ItemReplicates

Test Item Concentration

Repl.

Oxygen
Concentration*

Oxygen Uptake Rate R

Inhibition

Mean Inhibition

[mg/L]

 

[mg O2/L]

[mg O2/(L×h)]

[%]

 [%]

1000

1

7.56

34.9

-5

-5

2

7.19

35.6

-7

3

6.85

34.0

-2

*) determined at start of the measurement

Repl. = Replicate

Validity criteria fulfilled:
yes
Conclusions:
The NOEC of the test item N,N'-[(methylimino)bis(trimethylene)]bis(stearamide) is ≥ 1000 mg/L.
Executive summary:

For N,N’-[(methylimino)bis(trimethylene)]bis(stearamide) a respiration Inhibition Test with activated sludge according to OECD Guideline No. 209 (2010) was carried out.

A preliminary test with 10, 100, 1000 mg/l showed no relevant inhibiting of the heterotrophic respiration or nitrification (tested with Allylurea ATU).

The main test was conducted as limit test under static conditions with the limit test item concentration of 1000 mg/L.

The respiration rates of the control, reterence and test item replicates were measured after a contact time of three hours, and the inhibitory effects of the test and reference item were determined in comparison to the control respiration rates. The mean inhibition of respiration for the test item replicates was - 5%.

The NOEC for N·N'-[(methylimino)bis(trimethylene)]bis(stearamide) was 1000 mg/L ( No statistically significant inhibiting effect (P = 0.021)).

The validity criteria of the test guideline were fulfilled.

Description of key information

For N,N’-[(methylimino)bis(trimethylene)]bis(stearamide) a respiration Inhibition Test with activated sludge according to OECD Guideline No. 209 (2010) was carried out.

A preliminary test with 10, 100, 1000 mg/l showed no relevant inhibiting of the heterotrophic respiration or nitrification (tested with Allylurea ATU).

The main test was conducted as limit test resulting in a 3h-NOEC=1000 mg/l.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
1 000 mg/L

Additional information

For N,N’-[(methylimino)bis(trimethylene)]bis(stearamide) a respiration Inhibition Test with activated sludge according to OECD Guideline No. 209 (2010) was carried out.

A preliminary test with 10, 100, 1000 mg/l showed no relevant inhibiting of the heterotrophic respiration or nitrification (tested with Allylurea ATU).

The main test was conducted as limit test under static conditions with the limit test item concentration of 1000 mg/L.

The respiration rates of the control, reterence and test item replicates were measured after a contact time of three hours, and the inhibitory effects of the test and reference item were determined in comparison to the control respiration rates. The mean inhibition of respiration for the test item replicates was - 5%.

The 3h-NOEC for N·N'-[(methylimino)bis(trimethylene)]bis(stearamide) was 1000 mg/L ( No statistically significant inhibiting effect (P = 0.021)).

The validity criteria of the test guideline were fulfilled.

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