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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-11-28 to 2012-11-29
Reliability:
1 (reliable without restriction)
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
GLP compliance:
yes
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 treated with a mortar prior to weighing. Afterwards all test item concentrations were weighed out on an object slide and given in the Erlenmeyer flasks, filled up with 200 mL demineralised water and stirred overnight.
- Eluate: Synthetic waste water acc. to OECD 209
- Differential loading: 1000 mg/L
- Controls: Triplicates at the beginning of the test + triplicates at the end of the test
Test organisms (species):
activated sludge
Details on inoculum:
- Preparation of inoculum for exposure: Non adapted activated sludge from the sewage plant at Hildesheim (Municipal sewage treatment plant of 31137 Hildesheim, Germany)
- Pretreatment: Washed twice and was used within 24 h after sampling
- Dry sludge concentration: 3.28 g/L corresponding to 1.64 g/L in the test vessel
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Hardness:
Not determined
Test temperature:
20.8 °C
pH:
pH-value of the activated sludge 7.47
pH-value of the synthetic waste water 7.50
Salinity:
Not determined
Details on test conditions:
TEST SYSTEM
- Test vessel: 1000 mL Erlenmeyer flask
- Aeration: Shaking of flasks at 150 rpm to keep the dissolved oxygen concentration above 60 - 70 % saturation and to maintain
the sludge flakes suspension.
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
- Composition of test medium:
250 mL inoculum
16 mL synthetic waste water
234 mL demineralised water



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
After 3 h incubation, the oxygen concentration of each control, reference item and test item replicate was determined by means of oximeter immediately after transferring the solution to the measuring cell and the oxygen depletion was recorded for at least 3 minutes on a flat bed recorder.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: None- limit test item concentration
- Range finding study
Inhibition of Respiration (Preliminary Tests)
Nominal Test Item
Concentration
[mg/L] Pretreatment pH(test item in water) Inhibition[%]
10 None - -2
100 - -4
1000 - -5
1000 Stirring overnight 7.44 1






Reference substance (positive control):
yes
Remarks:
Copper (II) sulphate pentahydrate p.a.
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
Remarks:
respiration rate
Details on results:
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- Relevant effect levels: EC50: 91.4 mg/L
Reported statistics and error estimates:
The NOEC was determined by calculation of statistical significance of the inhibition of respiration in comparison to the control. One Way Analysis of Variance (ANOVA) was used for NOEC calculation. When running a One Way Analysis of Variance a Normality test and an Equal Variance test were done first. P-value for both tests was 0.05. The a-value (acceptable probability of incorrectly concluding that there is a difference) is a=0.05.
The EC-value of the reference item was calculated by linear regression using software GraphPad Prism. Calculation of the confidence intervals for the EC50-value was carried out using standard procedures.
The coefficient of variation of the oxygen uptake rates of the control replicates was calculated with standard methods using Excel.

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 and reference item concentrations in % was calculated in comparison to the oxygen uptake rates of the control.

Oxygen Uptake Rates, Specific Respiration Rates and Inhibition of the Respiration
of Control, Test and Reference Item Concentrations

Test Item and Reference Concentration

[mg/L]

Repl.

Oxygen Uptake Rate
R
[mg O2/L
×h]

Specific Respiration Rate
Rs
[mg O2/g
×h]

Inhibition


[%]

Mean Inhibition

[%]

Control

1

42.4

25.9

-

-

2

42.4

25.9

3

40.4

24.6

4

35.2

21.5

5

35.2

21.5

6

35.2

21.5

Test item
1000

1

33.6

13

12

2

33.6

13

3

34.4

11

Reference item
58

1

26.4

31

32

2

26.4

31

3

25.2

35

100

1

18.0

53

53

2

18.0

53

3

18.0

53

180

1

9.2

76

77

2

8.4

78

3

8.8

77

NOEC and EC-Values of Fatty acids, C16-18-, reaction products with diethanolamine

EC-values
[mg/L]

NOEC*

1000

EC20

> 1000

EC50

> 1000

EC80

> 1000

Validity criteria fulfilled:
yes
Conclusions:
The NOEC of the test item Fatty acids, C16-18-, reaction products with diethanolamine is
1000 mg/L. Fatty acids, C16-18-, reaction products with diethanolamine caused no inhibiting
effects on activated sludge of a municipal sewage treatment plant at the limit concentration of 1000 mg/L.
Executive summary:

A Respiration Inhibition Test with activated sludge according to OECD Guideline No. 209 (2010) was carried out with the test item Fatty acids, C16-18-, reaction products with diethanolamine (batch number: ITA0058330) from 2012-11-28 to 2012-11-29; with the definitive exposure on 2012-11-29 at Dr.U.Noack-Laboratorien, 31157 Sarstedt, Germany. Test system was activated sludge of the municipal treatment plant of 31137 Hildesheim, Germany. The test was carried out under static conditions with the limit concentration 1000 mg/L. The respiration rates of the control, reference 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 inhibition of respiration for the test item replicates ranged from 11 to 13 % (Table 3).

In order to check the activity of the test system and the test conditions a reference test was carried out with copper (II) sulphate pentahydrate as reference item and the reference toxicity was determined. The EC50-value for the reference item was 91.4 mg/L.

The validity criteria of the test guideline were fulfilled.

Table 1:           NOEC and EC-Values of Fatty acids, C16-18-, reaction products with diethanolamine

EC-values
[mg/L]

NOEC*

1000

EC20

> 1000

EC50

> 1000

EC80

> 1000

                         *) No statistically significant effect (p 0.05), ANOVA

The NOEC of the test item Fatty acids, C16-18-, reaction products with diethanolamine is 1000 mg/L. Fatty acids, C16-18-, reaction products with diethanolamine caused no inhibiting effects on activated sludge of a municipal sewage treatment plant at the limit concentration of 1000 mg/L.

Description of key information

The NOEC of the test item Fatty acids, C16-18-, reaction products with diethanolamine is

1000 mg/L. Fatty acids, C16-18-, reaction products with diethanolamine caused no inhibiting

effects on activated sludge of a municipal sewage treatment plant at the limit concentration of 1000 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
1 000 mg/L

Additional information

A Respiration Inhibition Test with activated sludge according to OECD Guideline No. 209 (2010) was carried out with the test item Fatty acids, C16-18-, reaction products with diethanolamine (batch number: ITA0058330) from 2012-11-28 to 2012-11-29; with the definitive exposure on 2012-11-29 atDr.U.Noack-Laboratorien, 31157 Sarstedt, Germany.Test system was activated sludge of the municipal treatment plant of 31137 Hildesheim, Germany. The test was carried out under static conditions with the limit concentration 1000 mg/L. The respiration rates of the control, reference 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 inhibition of respiration for the test item replicates ranged from 11 to 13 % (Table 3).

In order to check the activity of the test system and the test conditions a reference test was carried out with copper (II) sulphate pentahydrate as reference item and the reference toxicity was determined. The EC50-value for the reference item was 91.4 mg/L.

The validity criteria of the test guideline were fulfilled.

Table 1:           NOEC and EC-Values of Fatty acids, C16-18-, reaction products with diethanolamine

EC-values
[mg/L]

NOEC*

1000

EC20

> 1000

EC50

> 1000

EC80

> 1000

                        *) No statistically significant effect (p 0.05), ANOVA

The NOEC of the test itemFatty acids, C16-18-, reaction products with diethanolamine is1000mg/L. Fatty acids, C16-18-, reaction products with diethanolamine caused no inhibiting effects on activated sludge of a municipal sewage treatment plant at the limit concentration of 1000 mg/L.