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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-04-25 - 2016-05-03 (experimental phase)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
Version / remarks:
OECD Test Guideline No. 209 in the Version dated 22-Jul-2010
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Hess. Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Mainzer Straße 80, D65189 Wiesbaden
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: direct weighing
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): none stated
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Laboratory culture:
- Name and location of sewage treatment plant where inoculum was collected: derived from the effluent of the aeration tank of the municipal sewage treatment plant at Taunusstein-Bleidenstadt, Germany, dealing with pre-dominantly domestic sewages.
- Method of cultivation: This synthetic sewage used for the test and for feeding procedures was prepared according to the OECD test guideline 209. Immediately after receipt in the laboratory the activated sludge was washed twice with mineral nutrient medium acc. OECD 301B and aerated thereafter until test start. 10 L of the washed sludge were fed with 500 mL of the synthetic sewage. The feeding procedure was repeated daily.
- Initial biomass concentration: The dry mass of the activated sludge determined one day before start of the main test was 4.31 g/L. A theoretical dry matter content of 3.75 g/L should be achieved by the addition of 1490 mL tap water/10 L sludge. The dry matter content was controlled after completion of the test in order to calculate the respiration rates with the precisely measured dry matters. As a result of that control it must be stated that the dry matter content was slightly lower than the theoretical value: the final dry matter content was 3.442 g/L resulting in a final concentration of 1.38 g/L of dry mass in the test treatments which is in accordance with the indications of 1.5 g/L ± 10% given in the study plan. The results are based on the actual final dry matter content.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Remarks on exposure duration:
In order to get the same incubation period of 3 hr for each test solution, a new test solution was started every 5 minutes.
Post exposure observation period:
none
Test temperature:
20±2°C
pH:
7.7 - 8.7
Dissolved oxygen:
aerated
Nominal and measured concentrations:
0, 125, 250, 500, 1000, 2000 mg/L (nominal)
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL glass bottles
- Type (delete if not applicable): open / closed
- Material, size, headspace, fill volume: 250 ml fill volume
- Aeration: Yes, Aeration device: wetted compressed air from central system equipped with a system of tubings and single-use Pasteur pipettes
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 5
- No. of vessels per abiotic control (replicates): 6
- Sludge concentration (weight of dry solids per volume): final concentration of 1.38 g/L of dry mass
- Nitrification inhibitor used (delete if not applicable): N-allylthiourea

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: deionised water

OTHER TEST CONDITIONS
- Adjustment of pH: no

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : respiration rate
TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study
- Test concentrations: 0, 10, 100, 1000 mg/l
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
307 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Duration:
3 h
Dose descriptor:
other: EC20
Effect conc.:
527 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
1 487 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
< 136.56 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Duration:
3 h
Dose descriptor:
LOEC
Effect conc.:
136.56 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
449 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of heterotrophic respiration
Duration:
3 h
Dose descriptor:
other: EC20
Effect conc.:
757 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of heterotrophic respiration
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
2 057 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of heterotrophic respiration
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
258.88 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of heterotrophic respiration
Duration:
3 h
Dose descriptor:
LOEC
Effect conc.:
511.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of heterotrophic respiration
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
510.64 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of respiration due to nitrification
Remarks on result:
other:
Remarks:
No distinct Effect concentration can be given due to the lack of a concentration / response. Absolute nitrification rates were extremely low.
Duration:
3 h
Dose descriptor:
LOEC
Effect conc.:
1 011.9 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of respiration due to nitrification
Remarks on result:
other:
Remarks:
No distinct Effect concentration can be given due to the lack of a concentration / response. Absolute nitrification rates were extremely low.
Details on results:
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: none stated
- Effect concentrations exceeding solubility of substance in test medium: none, water solubility was determined as 3.65 g/L which is above all noted effect concentrations
- Adsorption (e.g. of test material to the walls of the test container): none stated
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: The EC50 of 3,5-Dichlorophenol for total respiration was in the required range of 2-25 mg/L since an inhibition of 58 % occurred at 30 mg/L.

Results

 

Screening-Test

 

Respiration Rates and Inhibition Values within the Screening-Test

 

Concentration[mg/L]

Respiration Rate
Total(- ATU)
mg O2/g DM * h


Respiration Rate
 (+ ATU)
mg O2/g DM * h
heterotrophic

Calculated Respiration Rate due to Nitrification

mg O2/g DM * h

% Inhibition of the Total Respiration Rate

% Inhibition of the hetero-trophic Respiration Rate

% Inhibition of the Nitrification Rate*)

Control

17,90

19,00

0,03

-

-

-

19,80

19,30

-0,27

17,60

19,40

-0,37

20,80

17,70

1,33

10

17,50

19,00

0,03

8,0

-0,8

85,7

100

17,7

18,2

0,83

7,0

3,4

-371,4

1000

12,2

11,8

7,23

35,9

37,4

-4028,6

 

DM = Dry mass; ATU = allylthiourea; *) Negative value indicates an activation (increase of the respiration rate); There was no autoxidation at the highest concentration of 1000 mg/L. Thus, the respiration rates were not corrected.

 

 

pH Values and Temperatures in the Treatments within the Screening-Test

Concentration of the Test Item [mg/L]

Temperature [°C] after Incubation

pH at Beginning of the Incubation

pH after Incubation

Control (0) without ATU

19.2

7.78

8.53

19.3

7.74

8.38

19.0

7.75

8.57

19.7

7.59

8.37

Control (0) with ATU

19.5

7.77

8.44

19.5

7.75

8.45

19.3

7.75

8.45

19.3

7.75

8.55

10 without ATU

19.4

7.75

8.43

10 with ATU

19.4

7.75

8.46

100 without ATU

19.3

7.75

8.48

100 with ATU

19.2

7.75

8.47

1,000 without ATU

19.6

7.75

8.45

1,000 with ATU

19.8

7.77

8.49

 

 

 

Definitive-Test

 

Dichlorophenol-Reference

 

Results of the Dichlorophenol-Reference

Concentration of the Reference Item 3,5-Dichlorophenol [mg/L]

Temperature [°C] after Incubation

pH at Beginning of the Incubation

pH after

Incubation

Respiration Rate (mg O2/g DM*hr) after 3hr of Inc.

Inhibition of Respiration Rate [%]

5

19.1

7.72

8.55

22.4

-4.9

15

19.0

7.77

8.58

11.9

44.3

30

19.0

7.75

8.61

8.9

58.3

 

 

Test Item

 

Respiration Rates and Inhibition Values within the Definitive Test

Concentration[mg/L]

Respiration Rate
Total(- ATU)
mg O2/g DM * h

Respiration Rate
 (+ ATU)
mg O2/g DM * h
heterotrophic

Calculated Respiration Rate due to Nitrification

mg O2/g DM * h

% Inhibition of the Total Respiration Rate1)

% Inhibition of the heterotr. Respiration Rate1)

% Inhibition of the Nitrification Rate1)

Control

20.6

19.9

1.5

-

-

-

20.2

21.0

0.3

22.0

20.2

1.2

21.0

15.3

6.1

18.9

17.7

3.7

25.4

23.2

-1.9

mean

21.35

19.55

1.80

1252)

18.7

20.0

-0.4

12.4

-2.3

121.1

20.4

19.4

0.2

4.4

0.8

87.8

19.8

18.9

0.7

7.3

3.3

60.0

20.5

18.9

0.7

4.0

3.3

60.0

18.7

19.1

0.5

12.4

2.3

71.1

mean

19.6

19.3

0.4

8.1

1.5

80.5

2502)

19.6

20.2

-0.3

8.2

-3.3

114.4

19.8

19.5

0.4

7.3

0.3

75.6

20.2

19.7

0.2

5.4

-0.8

86.7

20.3

19.8

0.1

4.9

-1.3

92.2

19.8

19.2

0.7

7.3

1.8

58.9

mean

19.9

19.7

0.3

6.6

-0.7

85.6

5002)

18.8

17.5

0.5

11.9

10.5

74.4

18.6

16.9

1.1

12.9

13.6

41.1

18.0

16.7

1.3

15.7

14.6

30.0

17.1

14.4

3.6

19.9

26.3

-97.8

17.3

16.3

1.7

19.0

16.6

7.8

mean

18.0

16.4

1.6

15.9

16.3

11.1

10002)

12.2

15.7

-3.1

42.9

19.7

271.1

11.0

15.2

-2.6

48.5

22.3

243.3

11.2

13.1

-0.5

47.5

33.0

126.7

14.6

15.3

-2.7

31.6

21.7

248.9

14.1

13.6

-1.0

34.0

30.4

154.4

mean

12.6

14.6

-2.0

40.9

25.4

208.9

20002)

9.5

9.2

0.6

55.5

58.1

66.7

8.6

9.6

-0.8

59.7

50.9

144.4

9.0

8.8

0.0

57.8

55.0

100.0

8.8

10.7

-1.9

58.8

45.3

205.6

8.1

12.0

-3.2

62.1

38.6

277.8

mean

8.8

9.9

-1.1

58.8

49.6

158.9

 

DM = Dry mass; ATU = allylthiourea;1)Negative value indicates an activation (increase of the respiration rate);*)significant inhibition;2)exact concentrations have to be indicated for each individual treatment as the test item was weighed individually into the treatments separately.

 

Calculated Individual Concentrations in the Treatments

Nominal Conc.

125 mg/L

500 mg/L

 

 

Weight in mg/250 mL

Weight in mg/250 mL

 

 

- ATU (a)

+ ATU (b)

- ATU (a)

+ ATU (b)

 

 

33.1

34.5

125.9

126.8

 

34.4

34.2

128.5

126.5

 

33.8

40.9

125.5

127.6

 

34.1

39.5

130.3

133.3

 

35.3

37.5

126.9

125.3

 

Mean:

34.14

37.32

127.4

127.9

 

Final mean Conc.:

136.56

149.28

509.6

511.6

 

Mean of (a) and (b):

142.92 mg/L

510.64 mg/L

 

Nominal Conc.

250 mg/L

1000 mg/L

2000 mg/L

 

65.3

63.8

252.8

250.7

507.7

500.3

66.1

65.0

254.1

251.3

500.0

500.0

68.9

65.2

255.8

251.8

504.4

504.7

64.8

64.1

250.4

252.6

501.8

502.6

64.0

65.5

258.1

252.2

500.0

502.6

Mean:

65.82

64.72

254.2

251.72

502.78

502.04

Final mean Conc.:

263.28

258.88

1016.96

1006.88

2011.12

2008,16

Mean of (a) and (b):

261.08 mg/L

1011.92 mg/L

2009.64 mg/L

 

pH Values and Temperatures in the Treatments within the Definitive Test

 

Concentration of the Test Item [mg/L]

Temperature [°C] after Incubation

pH at Beginning of the Incubation

pH after Incubation

 

 

Control (0) without ATU

19.5

7.70

8.49

 

 

19.3

7.75

8.61

 

 

19.5

7.73

8.46

 

 

19.0

7.79

8.58

 

 

19.3

7.84

8.65

 

 

21.0

7,79

8.55

 

 

Control (0) with ATU

19.4

7.72

8.60

 

 

19.3

7.77

8.54

 

 

19.2

7.30

8.56

 

 

19.0

7.79

8.66

 

 

19.0

7.84

8.66

 

 

20.8

7.82

8.58

 

 

125 mg/L without ATU

19.2

7.78

8.51

 

 

19.2

7.78

8.58

 

 

19.3

7.78

8.54

 

 

19.5

7.78

8.45

 

 

19.1

7.78

8.60

 

 

125 mg/L with ATU

19.3

7.77

8.45

 

 

19.0

7.79

8.55

 

 

19.3

7.80

8.53

 

 

19.4

7.80

8.47

 

 

19.6

7.80

8.53

 

 

250 mg/L without ATU

19.4

7.78

8.55

 

 

19.3

7.80

8.55

 

 

19.4

7.77

8.58

 

 

19.5

7.77

8.59

 

 

19.3

7.77

8.62

 

 

250 mg/L with ATU

19.5

7.71

8.56

 

 

19.4

7.71

8.59

 

 

19.5

7.70

8.52

 

 

19.5

7.70

8.50

 

 

19.5

7.70

8.55

 

 

500 mg/L without ATU

19.4

7.73

8.51

 

 

19.4

7.73

8.51

 

 

19.3

7.76

8.48

 

 

19.2

7.76

8.55

 

 

19.1

7.76

8.54

 

 

500 mg/L with ATU

19.1

7.77

8.46

 

 

19.0

7.78

8.57

 

 

19.0

7.77

8.56

 

 

19.0

7.77

8.68

 

 

19.0

7.79

8.61

 

1000 mg/L without ATU

19.0

7.80

8.57

19.0

7.78

8.61

19.0

7.80

8.61

19.1

7.81

8.61

19.3

7.81

8.61

1000 mg/L with ATU

19.7

7.82

8.58

19.7

7.81

8.63

19.2

7.84

8.67

19.7

7.81

8.58

19.4

7.83

8.64

2000 mg/L without ATU

19.4

7.84

8.64

19.0

7.85

8.67

19.2

7.83

8.66

19.1

7.85

8.63

19.0

7.85

8.70

2000 mg/L with ATU

19.0

7.85

8.50

19.0

7.84

8.58

19.0

7.84

8.63

20.2

7.83

8.55

20.6

7.83

8.60

 

 

Effective Concentrations

 

Under the conditions of the test the following EC-values were determined:

 

Inhibition of the Total Respiration

 

EC10    = 307 mg/L [121.6 – 468.7] *)

EC20    = 527 mg/L [293.1 – 716.3] *)

- EC50    = 1487 mg/L [1141.5 – 2229.5] *)

NOEC < 136.56 mg/L

LOEC= 136.56 mg/L

 

*) values in brackets indicate the 95% confidence level

 

Inhibition of the heterotrophic Respiration

 

EC10    =  449 mg/L [192.4 – 650.5] *)

EC20    =  757 mg/L [461.3 – 983.0] *)

EC50    = 2057 mg/L [1585.8 – 3357.0] *)

NOEC = 258.88 mg/L

LOEC= 511.60 mg/L

 

*) values in brackets indicate the 95% confidence level

 

Inhibition of the Respiration due to Nitrification

 

No distinct Effect concentration can be given due to the lack of a concentration / response. Absolute nitrification rates were extremely low.

NOEC =  510.64 mg/L

LOEC= 1011.90 mg/L

 

 

Criteria for Validity of the Results

 

All conditions of validity of the test were kept within this study:

The blank control (without the test item or reference item) oxygen uptake rates were higher than 20 mg/g dry mass of suspended solids in an hour.

The coefficient of variation of oxygen uptake in the control replicates was not higher than 30%.

The EC50of 3,5-Dichlorophenol for total respiration was in the required range of 2-25 mg/L since an inhibition of 58 % occurred at 30 mg/L.

Validity criteria fulfilled:
yes
Remarks:
All conditions of validity of the test were kept within this study.
Conclusions:
The study was conducted under GLP according to OECD 209 without deviations on the registered substance itself. The method is to be considered scientifically reasonable and suitable for the test item, the available information allows the conclusion that the test was properly conducted, the positive control showed the appropriate results, the validity criteria were all fulfilled. Hence, there is no doubt that the obtained results are not reliable. Based on an EC50 = 1487 mg/L (total respiration), it can be concluded that the substance is relatively harmless to the aquatic environment and when released into a sewage treatment plant.
Executive summary:

3-Methyl-thiazolidin-thion-2 was tested for toxicity towards activated sludge according to OECD Guideline 209 in the version dated 22-Jul-2010. For the determination of the toxic behaviour of the test item, activated sludge from the aeration tank of the municipal sewage treatment plant at Taunusstein-Bleidenstadt, Germany, was exposed to the nominal test item concentrations of up to 2000 mg/L together with and without the specific nitrification inhibitor allylthiourea. A toxic behaviour is demonstrated by the reduction of the respiration rates. After 3 hours of incubation period the inhibition of the respiration rates of the activated sludge were determined in comparison to the respective set of test solutions without any test item.

 

All conditions of validity of the test were kept within this study: The blank control (without any test or reference item) oxygen uptake rates were higher than 20 mg/g dry mass of suspended solids in an hour. The coefficient of variation of oxygen uptake in the control replicates was not higher than 30%. The EC50 of the reference item 3,5-Dichlorophenol for total respiration was in the required range of 2 to 25 mg/L.

Under the conditions of the test the following EC-values were determined:

 

Inhibition of the Total Respiration

EC10              = 307 mg/L [121.6 – 468.7] *)

EC20               = 527 mg/L [293.1 – 716.3] *)

EC50              = 1487 mg/L [1141.5 – 2229.5] *)

NOEC  < 136.56 mg/L

LOEC  = 136.56 mg/L

*) values in brackets indicate the 95% confidence level

 

Inhibition of the heterotrophic Respiration

EC10               =  449 mg/L [192.4 – 650.5] *)

EC20               =  757 mg/L [461.3 – 983.0] *)

EC50               = 2057 mg/L [1585.8 – 3357.0] *)

NOEC  = 258.88 mg/L

LOEC  = 511.60 mg/L

*) values in brackets indicate the 95% confidence level

 

Inhibition of the Respiration due to Nitrification

No distinct Effect concentration can be given due to the lack of a concentration / response. Absolute nitrification rates were extremely low.

NOEC  =  510.64 mg/L

LOEC  = 1011.90 mg/L

Description of key information

3-Methyl-thiazolidin-thion-2 was tested for toxicity towards activated sludge according to OECD Guideline 209 with nominal concentrations of up to 2000 mg/L together with and without the specific nitrification inhibitor allylthiourea for 3 h.

Under the conditions of the test the following EC-values were determined:

 

Inhibition of the Total Respiration

EC10              = 307 mg/L [121.6 – 468.7] *)

EC20               = 527 mg/L [293.1 – 716.3] *)

EC50              = 1487 mg/L [1141.5 – 2229.5] *)

NOEC  < 136.56 mg/L

LOEC  = 136.56 mg/L

*) values in brackets indicate the 95% confidence level

 

Inhibition of the heterotrophic Respiration

EC10               =  449 mg/L [192.4 – 650.5] *)

EC20               =  757 mg/L [461.3 – 983.0] *)

EC50               = 2057 mg/L [1585.8 – 3357.0] *)

NOEC  = 258.88 mg/L

LOEC  = 511.60 mg/L

*) values in brackets indicate the 95% confidence level

 

Inhibition of the Respiration due to Nitrification

No distinct Effect concentration can be given due to the lack of a concentration / response. Absolute nitrification rates were extremely low.

NOEC  =  510.64 mg/L

LOEC  = 1011.90 mg/L

Key value for chemical safety assessment

EC50 for microorganisms:
1 487 mg/L
EC10 or NOEC for microorganisms:
307 mg/L

Additional information

Key values refer to the Inhibition of the Total Respiration, 307 mg/l is the EC10 value as the NOEC could have only been determined as < 136.56 mg/L.