Registration Dossier

Administrative data

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP test performed according to OECD guideline.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
- Name of test material : 3-NITRO-1 ,2,4-TRIAZOL-5-0NE (NTO)
- Substance type: energetic explosive
- Physical state: light green to white crystalline solid with no odor
- Purity ca.99%
Specific details on test material used for the study:
Physical state: Fine white crystals
- Analytical purity: 100%
- Purity test date: 18 October 2012
- Lot/batch No.:DDP12K0091-0002
- Expiration date of the lot/batch: 2022

Sampling and analysis

Analytical monitoring:
not required

Test solutions

Vehicle:
yes
Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:The test substance was sufficiently soluble in RO water to prepare an aqueous stock solution. Therefore at test initiation 5 g (nominally) of the test substance was weighed into a 2L volumetric flask containing RO water. The pH of this solution was measured and adjusted to 7.5 ± 0.5 with 5M NaOH and HCl.

- Controls:Single replicates of the reference inhibitor 3,5-dichlorophenol (3,5-DCP) were used at 3, 10 and 32 mg/L, as a positive control.

- Evidence of undissolved material (e.g. precipitate, surface film, etc): None test material fully in solution

Test organisms

Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Laboratory culture: A sample of activated sludge was obtained the day before the start of the test from Worlingworth Sewage Treatment Works (Suffolk, UK), which treats predominantly domestic waste. In the laboratory, the sample was maintained under aerobic conditions until required.
- Method of cultivation:On the day of collection, an aliquot (10 mL) of the activated sludge was filtered through a dried and preweighed Whatman GF/C filter paper, which was then dried again at approximately 105C for at least one hour, allowed to cool in a desiccator and reweighed. The mixed liquor suspended solids (MLSS) content of the activated sludge was then calculated. The sludge was concentrated to ca. 4 g suspended solid/L by settling the sludge and removing a portion of supernatant. Synthetic sewage (50 mL/L) was added to the stock of activated sludge and this was aerated overnight.
- Preparation of inoculum for exposure:On the day of the test, the MLSS content of the sludge was determined (in triplicate) and adjusted to 4 g/L by the addition of tap water. The pH and temperature of the sludge were also measured. Aliquots (200 mL) were then added to each mixture to give a final MLSS concentration of 1.6 g/L.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Post exposure observation period:
Following the exposure period, the aeration was stopped and the headspace of each bottle was flushed with nitrogen during the oxygen measurement phase. The instrument measured the amount of oxygen in the mixtures at one minute intervals for at least 15 minutes. The pH and temperature of the samples were measured at the start and end of the test.

Test conditions

Hardness:
No data
Test temperature:
The mixtures were incubated at a temperature of 20-/+ 2°C.
pH:
3,5-DCP stock solution, pH 7.48
Activated sludge, pH 6.93
NTO stock solution pH 7.48
Synthetic seage pH 7.40
Dissolved oxygen:
No data
Salinity:
No data
Nominal and measured concentrations:
Nominal concentrations of NTO: 10, 100, 1000 mg/L
Details on test conditions:
A Co-ordinated Environmental Services (CES) Ltd automated respirometer and associated software was used to monitor the oxygen consumed by the test and control mixtures following a 3-hour exposure phase.
Mixtures were incubated in 500 mL bottles fitted with an oxygen probe, a sinter to deliver air and a nitrogen gas nozzle. Aliquots of RO water, synthetic sewage and antifoam agent (silicone oil in water) were added to each bottle. Immediately prior to initiation of the instrument, the required volume of activated sludge was added to each bottle.
The prepared mixtures were aerated and stirred for three hours in a thermostatically-controlled water bath, using an aerator connected to a laboratory supply of oil-free compressed air (one litre/minute).
Reference substance (positive control):
yes
Remarks:
3,5 dichlorophenol

Results and discussion

Effect concentrations
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
ca. 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:
NTO did not significantly inhibit the respiration rates of the samples of activated sludge. Mean respiration rates were reduced by 12% at 1000 mg/L; however, this was not considered statistically significant or biologically relevant.
Consequently, the 20%, 50% and 80% Effect Concentrations (EC20, EC50 and EC80) for inhibition could not be calculated but must be >1000 mg/L, the highest concentration employed in this study.
The no observed effect concentration (NOEC) for NTO was 1000 mg/L.
Results with reference substance (positive control):
The mean specific respiration rate of the control cultures was 30.2 mgO2/gh with a coefficient of variation of 15.4%. Sludge respiration rates were progressively reduced in the presence of increasing concentrations of 3,5-DCP. The three-hour 50% effect concentration (EC50) for 3,5-DCP was calculated to be 10.9 mg/L.
These results show that the sample of activated sludge employed was sensitive to inhibition and that the test was valid.
Reported statistics and error estimates:
None

Any other information on results incl. tables

Table1           Dissolved oxygen concentrations, measurement times and specific respiration rates

Group

Nominal Conc. (mg/L)

Bottle No.

Initial measured
DO concentration

(mgO2/L)

Final measured DO concentration

(mgO2/L)

Measurement time

(minutes)

Specific respiration rate

(Rs mgO2/gh)

Mean Rs
(mgO2/gh)

% inhibition

Mean % inhibition

 

 

 

 

 

 

 

 

 

 

Control

-

1

6.39

2.64

4

35.2

30.2

-

-

2

6.63

2.58

5

30.4

3

5.56

2.53

4

28.4

4

6.37

2.63

6

23.4

5

5.65

2.06

4

33.7

Test
NTO

10

6

5.33

2.02

4

31.0

31.9

-3

-6

7

7.02

3.83

4

29.9

1

8

6.96

2.62

6

27.1

10

9

7.07

2.21

5

36.5

-21

10

7.04

2.38

5

35.0

-16

100

11

6.70

2.39

6

26.9

31.2

11

-3

12

6.51

2.03

5

33.6

-11

13

6.35

2.05

5

32.3

-7

14

6.47

2.29

5

31.4

-4

15

6.46

2.24

5

31.7

-5

1000

16

5.88

2.97

5

21.8

26.4

28

12

17

6.35

3.05

6

20.6

32

18

6.41

2.07

5

32.6

-8

19

6.43

2.40

5

30.2

0

20

6.68

2.36

6

27.0

11

3,5-DCP

3

21

7.54

2.75

7

25.7

-

15

-

10

22

7.92

3.69

10

15.9

47

32

23

7.52

5.87

10

6.2

80

 

 

 

 

 

 

 

 

 

 

DO = Dissolved Oxygen

The data presented were calculated using unrounded values stored in the computer database. Minor numerical differences may be observed in the respiration rate calculation if rounded values are used to calculate the data. This minor discrepancy is not considered to be significant.

The Coefficient of variation for the control group was 15.4%.

Negative inhibition indicates a respiration rate greater than that of the mean control respiration rate.

           


Table2           Temperature and pH measurements

Group

Nominal Conc. (mg/L)

Bottle No.

Temperature (°C)

pH

Initial

Final

Initial

Final

 

 

 

 

 

 

 

Control

-

1

20.5

20.5

7.22

7.78

2

20.6

20.5

7.22

7.75

3

20.5

20.5

7.21

7.68

4

20.5

20.6

7.21

7.73

5

20.5

20.6

7.21

7.70

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Test

NTO

10

6

20.5

20.6

7.22

7.68

7

20.5

20.8

7.23

7.76

8

20.6

20.8

7.21

7.74

9

20.5

20.7

7.22

7.78

10

20.5

20.7

7.22

7.82

 

 

 

 

 

 

 

 

 

 

 

 

100

11

20.5

20.6

7.23

7.81

12

20.5

20.5

7.23

7.78

13

20.5

20.5

7.24

7.84

14

20.5

20.5

7.24

7.78

15

20.5

20.6

7.26

7.77

 

 

 

 

 

 

 

 

 

 

 

 

1000

16

20.6

20.6

7.31

7.84

17

20.6

20.6

7.31

7.83

18

20.7

20.7

7.28

7.85

19

20.7

20.7

7.31

7.88

20

20.4

20.7

7.31

7.81

 

 

 

 

 

 

 

 

 

 

 

 

 

 

3,5-DCP

3

21

20.7

20.7

7.26

7.95

10

22

20.6

20.7

7.27

7.92

32

23

20.7

20.7

7.31

7.95

 

 

 

 

 

 

 

 

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The no observed effect concentration (NOEC) for NTO was 1000 mg/L. The 50% effect concentration (EC50) could not be calculated but this must be >1000 mg/L, the highest concentration tested.