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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-03-01
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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: 1330 mg/L was weighed out directly in Erlenmeyer flasks (1330 mg/L corresponds to 1000 mg/L corrected for the water content).
- Eluate: Synthetic waste water acc. to OECD 209
- Differential loadings: 1000 mg/L
- Controls: Test medium without test or reference item. Triplicates at the beginning of the test + triplicates at the end of the test
-Reference item: three concentrations within the concentration range 58 - 180 mg/L and a dilution factor of 1.8., Triplicates
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)
- Receipt: 2016-02-29
- 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.
- Dry sludge concentration: 3.22 g/L, corresponding to 1.61 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:
20.2 °C
pH:
pH-value of the activated sludge 7.54
pH-value of the synthetic waste water 7.27
Salinity:
Not determined
Details on test conditions:
TEST SYSTEM
- Test vessel: 500 mL Erlenmeyer flask, DIN 12380
- Aeration: Permanent, to keep the dissolved oxygen concentration above 60 - 70 % saturation and to maintain the sludge flakes in suspension.
- Pretreatment: None

- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6 (Triplicates at the beginning of the test + triplicates at the end of the test.)
-No. of vessels per reference item (replicates): 9 (three concentrations within the range 58 - 180 mg/L and a dilution factor 1.8,at the end of the test)
- Composition of test medium:

Test concentration [mg/L] 1330
(1000 corrected for water content)
Test Item [mg] 665
(500 corrected for water content)
Synthetic waste water [mL] 16
Demineralised water [mL] Filled up to 250 mL
Inoculum [mL] 250



Dilution Table for the Reference Item
Reference item concentration [mg/L] 58 100 180
Stock solution [µL]* 96.7 166.7 300
Synthetic waste water [mL] 16
Demineralised water [mL] Filled up to 250 mL
Inoculum [mL] 250
* Stock solution with 300g/L


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
Inhibition of Respiration (Preliminary Tests)

Inhibitions in the Preliminary Tests
A preliminary test (non GLP) was carried out with the following test item concentrations of Genamin DMG 75 :
10 - 100 - 1000 mg/L (corresponding to 7.52 – 75.2 – 752 mg/L corrected for water content) with two replicates each and additionally two replicates with 1000 mg/L to study the inhibition of the nitrification using Allylthiourea (ATU). The pH was adjusted in one replicate with the concentration of 10 mg/L, 100 mg/L, 1000 mg/L and 1000 mg/L (corresponding to 7.52 – 75.2 – 752 mg/L corrected for water content) with ATU.


Inhibitions in the Preliminary Tests

Nominal Test Item Concentration
[mg/L] pH

(test item in demin. water) pH

(adjusted to) pH

(test item in medium) Inhibition


[%]
1st preliminary test - with pH adjustment, direct aeration
10 (7.52*) 8.20 7.17# 7.19 2
8.20 - 7.30 -7
100 (75.2*) 9.20 7.09# 7.23 1
9.23 - 7.49 4
1000 (752*) 9.99 7.73# 7.42 1
9.99 - 9.09 3
1000 (752*) + ATU** 9.95 7.33# 7.45

14
9.99 - 9.14 2

*) corrected for water content
**) ATU = N-Allylthiourea
#) pH adjusted with HCl


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 (752*) + ATU** 1 14 -8
3 2 4
*) corrected for water content
**) ATU = N-Allylthiourea



Based on the results of the preliminary test it could be concluded that the heterotrophic respiration as well as the nitrification are not inhibited. Therefore the definitive study was carried out with the concentration of 1330 mg/L (corresponds to 1000 mg/L corrected for water content) in triplicates (limit test to determine the NOEC). The concentration was weighed out directly in the Erlenmeyer flasks.

A fresh sample of activated sludge taken from the sewage plant Hildesheim was used for the test procedure. It was washed twice and adjusted with chlorine free tap water if necessary to a dry sludge concentration of 3.0 g/L ± 10 %.

Triplicates of the control without test item was included at the beginning and at the end of the test.
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
Results with reference substance (positive control):
- Results with reference substance valid? Yes


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 calculations. When running a One Way Analysis of Variance a Normality test and an Equal Variance test were done first. P-values for both Normality and Equal Variance test are 0.05. The P-value (acceptable probability of incorrectly concluding that there is a difference) is P=0.05.

The EC-values of the test item and the reference item were calculated by sigmoidal dose-response regression and linear regression, respectively, using software GraphPadPrism. Calculations of the confidence intervals for the EC-values were carried out using standard procedures.

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 with the oxygen uptake rates of the control.

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

Test and Reference Item Concentration

[mg/L]

Repl.

Oxygen Uptake Rate
R
[mg O2/L
×h]

Inhibition


[%]

Mean Inhibition

[%]

Control

1

36.8

-

-

2

35.6

3

34.8

4

32.8

5

32.8

6

34.0

Test item
1330

1

34.4

0

0

2

34.8

-1

3

34.4

0

Reference item
58

1

25.2

27

28

2

25.2

27

3

24.4

29

100

1

17.2

50

49

2

18.0

48

3

18.0

48

180

1

10.0

71

71

2

10.0

71

3

10.4

70

Repl. = Replicate

Coefficient of variation of oxygen uptake rates of the control: 4.6%

Suspended solids in the test vessel: 1.61g/L

Specific Respiration Rates of the Control Replicates

 

 

Repl.

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

 

Control

1

22.9

2

22.1

3

21.6

4

20.4

5

20.4

6

21.1

Mean value

21.4

Repl. = Replicate

Suspended solids in the test vessel: 1.61 g/L

 

Validity criteria fulfilled:
yes
Conclusions:
The NOEC of the test item Genamin DMG 75 is ≥ 1000 mg/L (corrected for the water content).
Genamin DMG 75 caused no inhibiting effects on activated sludge of a municipal sewage treatment plant at the limit test item concentration of 1000 mg/L (corrected for the water content).


Executive summary:

A Respiration Inhibition Test with activated sludge according to OECD Guideline No. 209 (2010) was carried out with the test item Genamin DMG 75 on 2016-03-01 at the test facility. The 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 test item concentration 1330 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 mean inhibition of respiration for the test item replicates was 0 % .

 

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 104 mg/L. The validity criteria of the test guideline were fulfilled.

 

 NOEC of Genamin DMG 75

 

Test item Concentration
[mg/L]

NOEC*

≥ 1330
(≥ 1000**)

                        *) No statistically significant effect (P = 0.946), ANOVA

                        **) corrected for water content

 

 

The NOEC of the test item Genamin DMG 75 is
≥1000 mg/L (corrected for the water content).

 

Genamin DMG 75 caused no inhibiting effects on activated sludge of a
municipal sewage treatment plantat the limit test item concentration of
1000 mg/L (corrected for the water content).

Description of key information

The NOEC of the test item Genamin DMG 75  is ≥ 1000 mg/L (corrected for the water content).

Genamin DMG 75 caused no inhibiting effects on activated sludge of a municipal sewage treatment plant at the limit test item concentration of 1000 mg/L (corrected for the water content).

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
1 000 mg/L

Additional information