Registration Dossier

Administrative data

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-08-23 to 2011-08-24
Reliability:
1 (reliable without restriction)

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
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent

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: No vehicle, directly weighing of the stock solution (5 g/L) Due to practical reasons the stock solution was prepared one day before test start in demineralised water.
- Eluate: Demineralised water
- Differential loading: 1000 - 580 - 320 - 180 - 100 mg/L
- Controls: Test medium without test or reference substance. 6 replicates: Triplicates at the beginning of the test + triplicates at the end of the test

Test organisms

Test organisms (species):
activated sludge
Details on inoculum:
- Preparation of inoculum for exposure: Non adapted activated sludge from the sewage plant at sarstedt (Municipal sewage treatment plant of 31157 Sarstedt, Germany)
- Pretreatment: Washed twice with autoclaved tap water and adjusted to a dry weight concentration of 3 g/L +/- 10 %
- Dry sludge concentration: 2.97 g/L

Study design

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

Test conditions

Hardness:
Not determined
Test temperature:
21.9 °C
pH:
7.25 (activated sludge)
7.42 (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
[mg/L] Mean Inhibition[%]
10 7
100 9
1000 36













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

Results and discussion

Effect concentrationsopen allclose all
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
499 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: n.a.
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 860 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
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: 106 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 are 0.05. The -value (acceptable probability of incorrectly concluding that there is a difference) is a=0.05.

The EC-values of the test item was calculated by sigmoidal dose-response regression and the EC50-value of the reference item by linear regression using software GraphPad Prism5, GRAPHPAD SOFTWARE, INC.

Any other information on results incl. tables

EC-Values with Confidence Interval of 2,2’-(Cyclohexylimino)diethanol

EC-values

Confidence interval

[mg/L]

P = 95 [%]

[mg/L]

NOEC1)

580

EC20

> 1000

EC50

> 1000

EC80

> 1000

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

17.2

11.5

2

16.8

11.3

3

18.8

12.6

4

16.0

10.7

5

17.2

11.5

6

17.2

11.5

58

1

13.6

21

20

2

13.6

21

3

14.0

19

100

1

9.2

47

47

2

9.2

47

3

9.2

47

180

1

3.6

79

76

2

4.8

72

3

4.0

77

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

[%]

100

1

18.4

-7

-6

2

19.2

-12

3

16.8

2

4

18.4

-7

5

18.4

-7

180

1

18.0

-5

-1

2

17.6

-2

3

16.4

5

4

16.4

5

5

18.4

-7

320

1

17.6

-2

2

2

17.2

0

3

15.6

9

4

18.0

-5

5

15.6

9

580

1

16.8

2

7

2

15.2

12

3

16.8

2

4

15.2

12

5

16.4

5

1000

1

14.4

16

20

2

14.4

16

3

12.0

30

4

12.8

26

5

14.8

14

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The NOEC of the test item 2,2’-(Cyclohexylimino)diethanol is 580 mg/L.
The test item is not toxic at concentrations
Executive summary:

A Respiration Inhibition Test with activated sludge according to OECD Guideline No. 209 was carried out with the test item 2,2’-(Cyclohexylimino)diethanol (batch number: ESD0011452) from 2011-08-23 to 2011-08-24, with the experimental phase on 2011-08-24 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 100 - 180 - 320 - 580 - 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 mean inhibition of respiration for the test item replicates ranged from -6 % to 20 %.

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 106 mg/L.

Validity criteria of the test guideline were fulfilled.

EC-Values with Confidence Interval of 2,2’-(Cyclohexylimino)diethanol

EC-values

Confidence interval

[mg/L]

P = 95 [%]

[mg/L]

NOEC1)

580

EC20

> 1000

EC50

> 1000

EC80

> 1000

                                               1) no statistically significant effect (p 0.05) , ANOVA, Dunnett`s Method