Registration Dossier

Administrative data

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-04-19 to 2012-04-20
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study with GLP and no defiencies

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
yes
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Diethyleneglycoldibutylether
- Molecular formula: C12H26O3
- Molecular weight: 218.34 g/mol
- Physical state: Liquid, colourless
- Stability under test conditions: Stable
- Storage condition of test material: Room temperature, protected from light, in original container

Sampling and analysis

Analytical monitoring:
no

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test item was weighed out directly in an Erlenmeyer flask, filled up with 200 mL demieralised water.
- Eluate: Test medium:
250 mL inoculum
16 mL synthetic waste water
234 mL autoclaved tap water
- Differential loading: 125 - 250 - 500 - 1000 - 2000 mg/L (test item)
- Controls: Test medium without test or reference item.

Test organisms

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 with autoclaved tap water and adjusted to a dry weight concentration of 3 g/L +/- 10 %. The sludge was used within 24 h after stirring.
- Dry sludge concentration: 3.12 g/L corresponding to 1.56 g/L suspended solids in the test vessels

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h

Test conditions

Hardness:
Not determined
Test temperature:
21.1 °C
pH:
7.32 (activated sludge)
7.56 (synthetic waste water)
Salinity:
None, freshwater
Details on test conditions:
TEST SYSTEM
- Test vessel: 1000 mL Erlenmeyer flask, DIN 12380
- Aeration: Permanent
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 3
- Composition of test medium:
250 mL inoculum
16 mL synthetic waste water
234 mL autoclaved tap water
Test or reference item concentration (directly weighed into each test vessel)


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
- Range finding study
Inhibition of Respiration (Preliminary Test)
Nominal Test Item Concentration pH pH Inhibition [%]
[mg/L] (test item in water) (test medium)
80 7.53 7.48 -8
400 7.54 7.54 9
2000 7.57 7.64 80
10000 7.60 7.66 91
















Reference substance (positive control):
yes
Remarks:
Copper (II) sulphate pentahydrate p.a.

Results and discussion

Effect concentrationsopen allclose all
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
769 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: 95% CI 691-852 mg/L
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
183 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: 95% CI <125-235 mg/L
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
125 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: n.a.
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: 71.0 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) and DUNNETT’s test 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 were 0.05. The -value (acceptable probability of incorrectly concluding that there is a difference) is =0.05.

The EC-values of the test item were calculated by sigmoidal dose-response regression and the EC50-value of the reference item by linear regression using software GraphPad Prism, GRAPHPAD SOFTWARE, INC. Calculation of the confidence intervals for the EC50-values was carried out using standard procedures.

The EC50-value of the reference item was calculated by linear regression using software GraphPad Prism5, GRAPHPAD SOFTWARE, INC.

The coefficient of variation of the oxygen uptake rates of the control replicates was calculated with standard methods using Excel, MICROSOFT CORPORATION.

Any other information on results incl. tables

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

Reference Item 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

25.6

16.4

2

23.2

14.9

3

29.2

18.7

4

28.4

18.2

5

29.2

18.7

6

28.8

18.5

58

1

16.4

40

42

2

16.8

39

3

14.8

46

100

1

8.8

68

64

2

10.8

61

3

10.0

64

180

1

4.4

84

87

2

3.6

87

3

2.8

90

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


Test item Concentration
[mg/L]


Repl.

Oxygen Uptake Rate
R
[mg O2/L
×h]


Inhibition

[%]

Mean Inhibition

[%]

125

1

23.6

14

8

2

22.4

18

3

27.6

-1

4

27.6

-1

5

24.8

9

250

1

24.8

9

12

2

25.2

8

3

24.0

12

4

22.4

18

5

23.6

14

500

1

19.6

28

35

2

15.6

43

3

15.2

45

4

18.8

31

5

20.0

27

1000

1

12.0

56

60

2

10.8

61

3

11.6

58

4

11.2

59

5

9.2

66

2000

1

3.6

87

84

2

5.2

81

3

4.0

85

4

4.0

85

5

4.4

84

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The NOEC of the test item is 125 mg/L, the EC10 183 mg/L and the EC50 769 mg/L
to activated sludge of a municipal sewage treatment plant.
Executive summary:

A Respiration Inhibition Test with activated sludge according to OECD Guideline No. 209 was carried out with the test item Diethyleneglycoldibutylether (batch number: DEG4136740) from 2012-04-19 to 2012-04-20, with the experimental phase on 2012-04-20 at Dr.U.Noack-Laboratorien, D-31157 Sarstedt. Test system was activated sludge of the municipal treatment plant of D-31137 Hildesheim. The test was carried out under static conditions with the nominal test item concentrations 125 - 250 - 500 - 1000 - 2000 mg/L (factor: 2). 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 ranged from 8 % to 84 %.

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 71.0 mg/L.

EC-Values and NOEC of Diethyleneglycoldibutylether

Effect values

Confidence interval

[mg/L]

P = 95 [%]

[mg/L]

NOEC

125

n.d.

EC10

183

125 - 235

EC20

326

282 - 370

EC50

769

691 – 852

EC80

1714

1503 - >2000

                                               n.d.= not determinable

The NOEC of the test item is 125 mg/L, the EC10 183 mg/L and the EC50 769 mg/L
to activated sludge of a municipal sewage treatment plant.