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Reference
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:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
Version / remarks:
22 July 2010
Deviations:
no
Principles of method if other than guideline:
In line with the guidance and due to effects seen in the range finder on nitrifying bacteria, total respiration, heterotrophic respiration and nitrification were tested.

The test item was stirred for 3 hours in order to test the degradation/transformation products. Based on prelimenary data 3 hours is equal to or greater than 6 degradation half-lives of the test item, which is generally sufficient to ensure that the test solution contains only degradation products. This is in accordance with the OECD Guidance Document on Aqueous-Phase Aquatic Toxicity Testing of Difficult Test Chemicals: Series on Testing and Assessment Document No. 23 (Second Edition, 2019) and ECHA Chapter R.7b: Endpoint specific guidance (v4.0, 2017) . This was supported by the hydrolysis study (OECD 111), which showed that hydrolysis at pH 7 and 9 was so rapid that it was not possible to measure the half lives, where measures were taken at least every 10 minutes (Determination of Physico-chemical Properties of N-Phenyl-diethanolamine, reaction products with formaldehyde, Petrovic, D, Study No. 20194879, 2020). The OECD 209 was conducted at pH 7.0 to 8.4. Further support for the fact only degradation products were tested comes from the OECD 201, 202 and 203 studies which showed the parent was completely degraded after 3 hours of stirring in environmentally relevant medium and that the concentration of the transformation/degradation products remained within 80-120 % of the concentration at time 0 hours, of the experiments (OECD 201 Augusiak, J.A., 2020; OECD 202 Augusiak, J.A., 2020; OECD 203 Augusiak, J.A., 2020).
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Confidential
- Purity, including information on contaminants, isomers, etc.: 100 %

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: N/A
- Specific activity: N/A
- Locations of the label: N/A
- Expiration date of radiochemical substance: N/A

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature protected from light (use of amber glassware of tinfoil wrap)
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: Stable under storage conditions. No solvent/vehicle used.
- Stability in the medium, i.e. sensitivity of the test material to hydrolysis and/or photolysis: Rapidly hydrolyses (< 1 hour ) in testing system as defined by OECD Series on testing and assessment document no. 23 (Second Ed. 2020) - degradates tested.
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: The test item was not sufficiently soluble to allow the preparation of a 10 g/L stock solution in water.
- Reactivity of the test material with the incubation material used (e.g. plastic ware): None

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): None
- Preliminary purification step (if any): None
- Final concentration of a dissolved solid, stock liquid or gel: N/A
- Final preparation of a solid (e.g. stock crystals ground to fine powder using a mortar and pestle): N/A

FORM AS APPLIED IN THE TEST (if different from that of starting material)
- Specify the relevant form characteristics if different from those in the starting material, such as state of aggregation, shape of particles or particle size distribution: N/A

INFORMATION ON NANOMATERIALS
- Chemical Composition: N/A
- Density: N/A
- Particle size & distribution: N/A
- Specific surface area: N/A
- Isoelectric point: N/A
- Dissolution (rate): N/A

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable)
- Description of the formulation, e.g. formulated product for foliar application; formulated product soil application; solution in organic solvent for soil application; formulated product seed treatment; solution in organic solvent seed treatment: N/A

OTHER SPECIFICS
- Other relevant information needed for characterising the tested material, e.g. if radiolabelled, adjustment of pH, osmolality and precipitate in the culture medium to which the test chemical is added: N/A
Analytical monitoring:
no
Details on sampling:
- Concentrations: N/A
- Sampling method: N/A
- Sample storage conditions before analysis: N/A
Not required in line with test guideline. Sufficient evidence that the degradation products will have been tested.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Mixtures were stirred in closed dark brown bottles for a short period of time in the combined limit/range finding test. In the final test these mixtures were stirred in closed dark brown bottles for at least 3 hours to ensure microorganisms were exposed to degradation products as the most relevant testing product. The 3 hours stirring was based on preliminary experiments during analytical verification which supported that 3 hours would be equal to or greater than 6 degradation half-lives of the test item, which is generally sufficient to ensure that the test solution contains only degradation products. This is in accordance with the OECD Guidance Document on Aqueous-Phase Aquatic Toxicity Testing of Difficult Test Chemicals: Series on Testing and Assessment Document No. 23 (Second Edition, 2019) and ECHA Chapter R.7b: Endpoint specific guidance (v4.0, 2017) . This was supported by the hydrolysis study (OECD 111), which showed that hydrolysis at pH 7 and 9 was so rapid that it was not possible to measure the half lives, where measures were taken at least every 10 minutes (Determination of Physico-chemical Properties of N-Phenyl-diethanolamine, reaction products with formaldehyde, Petrovic, D, Study No. 20194879, 2020). The OECD 209 was conducted at pH 7.0 to 8.4. Further support for the fact only degradation products were tested comes from the OECD 201, 202 and 203 studies which showed the parent was completely degraded after 3 hours of stirring in environmentally relevant medium and that the concentration of the transformation/degradation products remained within 80-120 % of the concentration at time 0 hours, of the experiments (OECD 201 Augusiak, J.A., 2020; OECD 202 Augusiak, J.A., 2020; OECD 203 Augusiak, J.A., 2020).

These mixtures were made for each exposure concentration separately, by direct addition of the test substance to the test system (vessel) in rescued light conditions.

- Eluate: N/A
- Differential loading: N/A
- Controls: Test medium without test item, treated in the same way as the test item solutions. Abiotic control: only test item without sludge. Nitrification control: test medium without test item but with nitrification inhibitor.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): N/A
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)):
- Test concentration separation factor: N/A
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): N/A
- Other relevant information: N/A
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Laboratory culture: No
- Name and location of sewage treatment plant where inoculum was collected: Municipal sewage treatment plant: 'Waterschap Aa en Maas', 's -Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.
- Method of cultivation: N/A
- Preparation of inoculum for exposure: The sludge was coarsely sieved (1 mm2) and allowed to settle. The supernatant was removed and ISO-medium was added. The concentration of the suspended solids was determined (3.0 g/L, as used for the test). The batch of sludge was used one day after collection. Therefore 50 mL of synthetic medium was added per litre of activated sludge at the end of the collection day. The sludge was kept aerated at test temperature until use.
- Pretreatment: N/A
- Initial biomass concentration: Not reported - suspended solids were 3 g/L.
Test type:
static
Water media type:
freshwater
Remarks:
Adjusted ISO-medium (freshwater) with synthetic sewage feed.
Limit test:
no
Total exposure duration:
3 h
Remarks on exposure duration:
In accordance with OECD 209 (2010) test guideline.
Post exposure observation period:
Not required.
Hardness:
Not reported
Test temperature:
19-20 °C
pH:
7.0 - 7.2 experiment start
8.1-8.4 experiment end
Dissolved oxygen:
> 5 mg/L at the start of the test continuous aeration and stirring throughout test.
Salinity:
Not reported.
Conductivity:
Not reported
Nominal and measured concentrations:
Combined limit/range finder (nominal concentrations): 0, 10, 100 and 1000 mg/L (without ATU), 1000 mg/L (with ATU), 1000 mg/L (abiotic control).

Main test (nominal concentrations): 0, 180, 320, 560, 1000 mg/L (with and without ATU).
Details on test conditions:
TEST SYSTEM
- Test vessel: 1-litre amber glass bottles
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: Test item solution (200 mL) and synthetic medium (16 mL) were mixed and made up to a final volume of 250 mL with Milli-RO water.
- Aeration: yes - continuous clean oil free air.
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): N/A
- No. of vessels per abiotic control (replicates): 1 (during combined limit/test range finder which showed no oxygen uptake and therefore was not executed during the main test)
- Sludge concentration (weight of dry solids per volume): 1.5 g/L suspended solid (dry weight basis) during testing
- Weight of dry solids per volume of reaction mixture per unit of time: N/A
- Nutrients provided for bacteria:
16 g Peptone
11 g meat extract
3 g urea
0.7 g NaCl
0.4 g CaCl2.2H2O
0.2 g MgSO4.7H2O
2.8 g K2HPO4
Dissolved in Milli-RO water, made up to 1 litre
and filtered. The pH was within 7.5 ± 0.5.

- Nitrification inhibitor used (delete if not applicable): N-allylthiourea (ATU) at 2.32 g/L (experiment was conducted with and without ATU)
- Biomass loading rate: N/A
TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
Adjusted ISO-medium, formulated using RO water (tap water purified by reverse osmosis; GEON Waterbehandeling, Berkel-Enschot, The Netherlands) with the following composition:
CaCl2.2H2O 211.5 mg/L
MgSO4.7H2O 88.8 mg/L
NaHCO3 46.7 mg/L
KCl 4.2 mg/L
- Particulate matter:
OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: N/A
- Light intensity: Not reported
- Details on termination of incubation:
EFFECT PARAMETERS MEASURED (with observation intervals if applicable): mg O2/L.h
TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8
- Justification for using fewer concentrations than requested by guideline: N/A
- Range finding study: Yes - prior to the final test a combined limit/range finder was conducted.
- Test concentrations: 0, 100, 180, 320, 560 and 1000 mg/L
- Results used to determine the conditions for the definitive study: Combined limit/range finder study and data gained from analytical method development on test item stability.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
180 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
test item was stirred for 3 hours and organisms were exposed to transformation/degradation products
Basis for effect:
inhibition of total respiration
Key result
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
134 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.:
529 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
test item was stirred for 3 hours and organisms were exposed to transformation/degradation products
Basis for effect:
inhibition of total respiration
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
180 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
test item was stirred for 3 hours and organisms were exposed to transformation/degradation products
Basis for effect:
inhibition of heterotrophic respiration
Key result
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
201 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
test item was stirred for 3 hours and organisms were exposed to transformation/degradation products
Basis for effect:
inhibition of heterotrophic respiration
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
602 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
test item was stirred for 3 hours and organisms were exposed to transformation/degradation products
Basis for effect:
inhibition of heterotrophic respiration
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
320 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
test item was stirred for 3 hours and organisms were exposed to transformation/degradation products
Basis for effect:
inhibition of nitrification rate
Key result
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
197 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
test item was stirred for 3 hours and organisms were exposed to transformation/degradation products
Basis for effect:
inhibition of nitrification rate
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
367 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
test item was stirred for 3 hours and organisms were exposed to transformation/degradation products
Basis for effect:
inhibition of nitrification rate
Details on results:
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No, however it should be noted the test was on the degradation products.
- Effect concentrations exceeding solubility of substance in test medium: No
- Adsorption (e.g. of test material to the walls of the test container): No
- Blank controls oxygen uptake rate: 26.88 and 21.36 mg O2/g.h for the experiment without and with ATU, respectively.
- Coefficient of variation of oxygen uptake rate in control replicates: 13.46 and 8.77 % for the experiment without and with ATU, respectively.
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- Relevant effect levels: Inaccordance with OECD 209 (2010)
EC50 of 3,5-DCP should lie in the range 2 mg/L to 25 mg/L for total respiration

Experimental EC50: 7.5 mg/L

EC50 of 3,5-DCP should lie in the range 5 mg/L to 40 mg/L for heterotrophic respiration

Experimental EC50: 20.5 mg/L

EC50 of 3,5-DCP should lie in the range 0.1 mg/L to 10 mg/L for nitrification

Experimental EC50: XX mg/L
- Other:
Reported statistics and error estimates:
ECx
For the reference item, calculation of the EC50 value was based on a 3-parameter normal cumulative distribution function (CDF) using non-linear regression analysis, with the percentages of respiration inhibition versus the logarithms of the corresponding concentrations of the reference item.
For the test item, calculation of the ECx value of the total respiration rate was based on Weibull analysis using linear maximum likelihood regression, with the percentages of respiration inhibition versus the logarithms of the corresponding concentrations of the test item. The calculation of the ECx values of the heterotrophic and nitrification respiration rate were based on a 3-parameter normal cumulative distribution function (CDF) using non-linear regression analysis, with the percentages of respiration inhibition versus the logarithms of the corresponding concentrations of the test item.

NOEC determination
An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the control revealed significant inhibition of the respiration rate (Williams Multiple Sequential t-test, α=0.05, one-sided, smaller).

Calculations were performed with ToxRat Professional v. 3.2.1 (ToxRat Solutions® GmbH, Germany).

The EC50 for total respiration was 529 mg/L (95% confidence interval: 483 - 578 mg/L).
The EC50 for heterotrophic respiration was 602 mg/L (95% confidence interval: 422 -
850 mg/L).
The EC50 for nitrification respiration was 367 mg/L (95% confidence interval: 118 -
1141 mg/L).

Table 1 - Final Test – Overview of Results

Concentration

(mg/L)

Mean respiration rate

% Inhibition of the respiration rate
(mean value)

(mg O2/L h)

(mg O2/g h)¹

Total respiration (T)

0

40.32

26.88

 

100

35.32

23.54*

12

180

37.48

24.98*

7.0

320

29.44

19.62*

27

560

16.37

10.91*

59

1000

9.22

6.15*

77

Heterotrophic respiration (H)

0

32.04

21.36

 

100

27.82

18.55

13

180

30.53

20.35

4.7

320

24.42

16.28*

24

560

14.86

9.91*

54

1000

9.65

6.44*

70

Nitrification respiration (N)

0

-

5.52

 

100

-

5.00

9.5

180

-

4.63

16

320

-

3.35

39

560

-

1.01*

82

1000

-

-0.29*

105

¹ The amount of suspended solids in the final test mixture was 1.5 g/L.

* Statistically significantly different compared to control.

Table 2 - Combined Limit/Range-Finding Test – Respiration Rate/Inhibition, pH Values

Replicate

Concentration

(mg/L)

pH

Respiration rate

% Inhibition

respiration rate

(mean value)

 

Start

End

(mg O2/L.h)

(mg O2/g.h)¹

 

C 1

0

7.6

7.6

44.78

29.85

 

 

C 2

0

7.6

7.1

43.33

28.89

 

 

C 3

0

7.6

7.1

44.53

29.69

 

 

C 4

0

7.6

7.1

50.85

33.90

 

 

C 5

0

7.6

7.2

37.17

24.78

 

 

C 6

0

7.6

7.5

40.34

26.89

 

 

C Mean

 

 

 

43.50

29.00 (RTB)

 

 

SD

 

 

 

4.62

3.08

 

 

CV (%)

 

 

 

11

11

 

 

 

 

 

 

 

 

 

CN 1

0

7.5

7.9

26.73

17.82

 

 

CN 2

0

7.5

8.0

24.05

16.03

 

 

CN Mean

 

 

25.39

16.93 (RHB)

 

 

R 1

1.0

7.6

8.0

33.90

22.60

22.07

 

R 2

3.2

7.6

8.2

16.25

10.83

62.64

 

R 3

10

7.6

8.1

9.67

6.45

77.77

 

R 4

32

7.6

8.1

2.38

1.59

94.53

 

 

 

 

 

 

 

 

 

T 1

10

7.6

7.6

44.63

29.75

-2.60

 

T 2

100

7.6

7.4

45.87

30.58

-5.45

 

T 3a

1000

7.5

7.3

7.24

4.83

83.36

 

T 3b

1000

7.5

7.4

12.50

8.33

71.26

 

T 3c

1000

7.6

7.4

8.18

5.45

81.20

 

T3 Mean

 

 

9.31

6.20*(RT)

78.61 (IT)

 

TN a

1000

7.5

7.4

9.91

6.61

60.97

 

TN b

1000

7.5

7.4

11.47

7.65

54.82

 

TN c

1000

7.5

7.3

8.40

5.60

66.92

 

TN Mean

 

 

9.93

6.62*(RH)

60.90 (IH)

 

TA

1000

7.6

7.4

0#

 

 

 

C:

Blank control

¹

The amount of suspended solids in the final test mixture was 1.5 g/L.

CN:

Nitrification control

RTB:

Total respiration in the blank

R:

Reference item, 3,5-dichlorophenol

RHB:

Heterotrophic respiration in the nitrification control

T:

Test item, N-Phenyl-diethanolamine, reaction products with formaldehyde

RT:

Total respiration with N-Phenyl-diethanolamine, reaction products with formaldehyde

TA:

Abiotic control of N-Phenyl-diethanolamine, reaction products with formaldehyde

RH:

Heterotrophic respiration with N-Phenyl-diethanolamine, reaction products with formaldehyde

TN:

N-Phenyl-diethanolamine, reaction products with formaldehyde withN-allylthiourea

IT:

% inhibition of total respiration relative to RTB

SD:

Standard deviation

IH:

% inhibition of heterotrophic respiration relative to RHB

CV:

Coefficient of variation

 

 

*

Statistically significantly different compared to control

#

No respiration, therefore expressed as 0 mg O2/L.h (see paragraph6.1)

Table 3 Final Test– Respiration Rate/Inhibition without ATU, pH Values


Replicate

Concentration

(mg/L)

pH

Respiration rate

% Inhibition

respiration rate

(mean value)

Start

End

(mg O2/L.h)

(mg O2/g.h)¹

C 1

0

7.1

8.1

40.50

27.00

 

C 2

0

7.1

8.2

30.13

20.09

 

C 3

0

7.2

8.2

42.52

28.35

 

C 4

0

7.1

8.3

41.36

27.57

 

C 5

0

7.1

8.2

40.98

27.32

 

C 6

0

7.1

8.2

46.40

30.93

 

C Mean

 

 

 

40.32

26.88

 

SD

 

 

 

5.43

3.62

 

CV (%)

 

 

 

13.46

13.46 (RTB)

 

 

 

 

 

 

 

 

R 1

1.0

7.1

8.2

33.82

22.55

16.11

R 2

3.2

7.1

8.3

28.65

19.10

28.93

R 3

10

7.2

8.3

18.43

12.29

54.29

R 4

32

7.2

8.3

6.77

4.51

83.21

 

 

 

 

 

 

 

T 1a

100.0

7.1

8.2

37.37

24.91

7.30

T 1b

100.0

7.1

8.2

35.17

23.45

12.76

T 1c

100.0

7.1

8.3

34.48

22.99

14.47

T 1d

100.0

7.1

8.3

31.93

21.29

20.80

T 1e

100.0

7.1

8.3

37.63

25.09

6.66

T 1 Mean

 

 

 

35.32

23.54*(RT)

12.40 (IT)

 

 

 

 

 

 

 

T 2a

180

7.1

8.3

37.06

24.71

8.07

T 2b

180

7.1

8.2

36.10

24.07

10.46

T 2c

180

7.1

8.2

38.68

25.79

4.06

T 2d

180

7.1

8.3

33.33

22.22

17.33

T 2e

180

7.1

8.2

42.21

28.14

-4.70

T 2 Mean

 

 

 

37.48

24.98*(RT)

7.04 (IT)

Table 3 cont.

T 3a

320

7.1

8.3

28.85

19.23

28.44

T 3b

320

7.1

8.3

30.57

20.38

24.17

T 3c

320

7.1

8.3

27.57

18.38

31.61

T 3d

320

7.1

8.3

29.24

19.49

27.47

T 3e

320

7.1

8.3

30.95

20.63

23.23

T 3 Mean

 

 

 

29.44

19.62*(RT)

26.98 (IT)

T 4a

560

7.1

8.2

14.33

9.55

64.45

T 4b

560

7.0

8.2

15.48

10.32

61.60

T 4c

560

7.0

8.2

16.08

10.72

60.11

T 4d

560

7.1

8.2

16.80

11.20

58.33

T 4e

560

7.1

8.2

19.14

12.76

52.52

T 4 Mean

 

 

 

16.37

10.91*(RT)

59.40 (IT)

T 5a

1000

7.0

8.2

8.17

5.45

79.73

T 5b

1000

7.0

8.2

9.60

6.40

76.19

T 5c

1000

7.0

8.2

12.70

8.47

68.50

T 5d

1000

7.0

8.2

8.12

5.41

79.86

T 5e

1000

7.0

8.2

7.50

5.00

81.40

T 5 Mean

 

 

 

9.22

6.15*(RT)

77.14 (IT)

Table 4 - Final Test– Heterotrophic Respiration Rate/Inhibition with ATU, pH Values


Replicate

Concentration

(mg/L)

pH

Respiration rate

% Inhibition

respiration rate

(mean value)

Start

End

(mg O2/L.h)

(mg O2/g.h)¹

CN1

0

7.2

8.4

32.85

21.90

 

CN2

0

7.2

8.4

28.33

18.89

 

CN3

0

7.2

8.3

35.39

23.59

 

CN4

0

7.2

8.4

30.71

20.47

 

CN5

0

7.2

8.4

30.05

20.03

 

CN6

0

7.2

8.4

34.89

23.26

 

CNMean

 

 

 

32.04

21.36 (RHB)

 

SD

 

 

 

2.81

1.87

 

CV (%)

 

 

 

8.77

8.77

 

 

 

 

 

 

 

 

RN1

1.0

7.2

8.3

38.66

25.77

-20.67

RN2

3.2

7.2

8.3

36.17

24.11

-12.90

RN3

10

7.2

8.2

26.08

17.39

18.59

RN4

32

7.2

8.4

10.26

6.84

67.97

 

 

 

 

 

 

 

TN1a

100.0

7.1

8.4

25.05

16.70

21.81

TN1b

100.0

7.1

8.3

32.38

21.59

-1.07

TN1c

100.0

7.1

8.4

28.77

19.18

10.20

TN1d

100.0

7.1

8.4

27.76

18.51

13.35

TN1e

100.0

7.1

8.4

25.14

16.76

21.53

TN1 Mean

 

 

 

27.82

18.55 (RH)

13.16 (IH)

 

 

 

 

 

 

 

TN2a

180

7.1

8.4

24.03

16.02

24.99

TN2b

180

7.1

8.4

31.14

20.76

2.80

TN2c

180

7.1

8.3

28.48

18.99

11.10

TN2d

180

7.1

8.3

29.59

19.73

7.64

TN2e

180

7.1

8.2

39.40

26.27

-22.98

TN2 Mean

 

 

 

30.53

20.35 (RH)

4.71 (IH)

Table 4 cont.

Replicate

Concentration

(mg/L)

pH

Respiration rate

% Inhibition

respiration rate

(mean value)

Start

End

(mg O2/L.h)

(mg O2/g.h)¹

TN3a

320

7.1

8.3

22.28

14.85

30.45

TN3b

320

7.1

8.3

21.93

14.62

31.55

TN3c

320

7.1

8.3

31.08

20.72

2.99

TN3d

320

7.1

8.3

22.51

15.01

29.74

TN3e

320

7.1

8.3

24.29

16.19

24.18

TN3 Mean

 

 

 

24.42

16.28*(RH)

23.78 (IH)

 

 

 

 

 

 

 

TN4a

560

7.0

8.2

14.75

9.83

53.96

TN4b

560

7.0

8.2

14.92

9.95

53.43

TN4c

560

7.0

8.2

11.54

7.69

63.98

TN4d

560

7.0

8.2

16.20

10.80

49.43

TN4e

560

7.0

8.2

16.88

11.25

47.31

TN4 Mean

 

 

 

14.86

9.91*(RH)

53.62 (IH)

 

 

 

 

 

 

 

TN5a

1000

7.0

8.1

9.45

6.30

70.50

TN5b

1000

7.0

8.1

10.20

6.80

68.16

TN5c

1000

7.0

8.1

9.81

6.54

69.38

TN5d

1000

7.0

8.1

10.57

7.05

67.01

TN5e

1000

7.0

8.2

8.24

5.49

74.28

TN5 Mean

 

 

 

9.65

6.44*(RH)

69.87 (IH)

CN:      Control nitrification

SD:     Standard deviation

CV:      Coefficient of variation

R:         Reference item, 3,5-dichlorophenol

T:         Test item, N-Phenyl-diethanolamine, reaction products with formaldehyde

¹:           The amount of suspended solids in the final test mixture was 1.5 g/L

RHB:     Total heterotrophic respiration in the control

RH:       Total heterotrophic respiration with Test Item

IH:        % inhibition of heterotrophic respiration relative to RC

Validity criteria fulfilled:
yes
Conclusions:
In conclusion, under the conditions of the present test N-Phenyl-diethanolamine, reaction products with formaldehyde was not toxic to waste-water bacteria (activated sludge) below a concentration of 180 mg/L and 320 mg/L for heterotrophic and nitrification respiration, respectively (NOEC). A NOEC for total respiration could not be determined, since significant inhibition of total respiration was observed at all test item concentrations. The results for inhibition of nitrification can be used to support the conclusion that the test item does not appear to have a specific inhibitory effect on nitrifying bacteria.
The EC50 for total respiration was 529 mg/L (95% confidence interval: 483 - 578 mg/L).
The EC50 for heterotrophic respiration was 602 mg/L (95% confidence interval: 422 -
850 mg/L).
The EC50 for nitrification respiration was 367 mg/L (95% confidence interval: 118 -
1141 mg/L).
Executive summary:

The objective of the study was to evaluate the test item for its ability to adversely affect aerobic microbial treatment plants and, if possible, to determine the EC50 and/or the no - observed effect concentration (NOEC). A final test was performed preceded by a combined limit/range-finding test. The study met the validity criteria prescribed by the study plan and was considered to be valid.

The test was conducted on the degradation products as the hydrolysis half-life of the test item was found to be < 1 hour. This was acheived by stirring the parent prior to microorganism exposure, for 3 hours (i.e. 6 times the half life).

This is in accordance with OECD document No. 23 (Second edition, 2019) and ECHA R.7b (v4.0, 2017).

 

The experiment was conducted with and without ATU in order to derive effects on the inhibition of total and heterotrophic respiration and nitrification. The experimental set up followed the OECD 209 guideline without deviation. Tested concentrations were 0, 180, 320, 560 and 1000 mg/L.

 

ECx and NOEC values were:

NOEC total respiration: 180 mg/L

NOEC heterotrophic respiration: 180 mg/L

NOEC nitrification: 320 mg/L

EC50 for total respiration was 529 mg/L (95% confidence interval: 483 - 578 mg/L).

EC50 for heterotrophic respiration was 602 mg/L (95% confidence interval: 422 - 850 mg/L).

EC50 for nitrification respiration was 367 mg/L (95% confidence interval: 118 - 1141 mg/L).

The test substance inhibited heterotrophic respiration and nitrification, the lowest EC10 was 134 mg/L for total respiration.

Description of key information

3 h EC50 for total respiration was 529 mg/L, heterotrophic respiration was 602 mg/L, nitrification respiration was 367 mg/L, test on activated sewage sludge microorganisms.  EC10 for total respiration was 134 mg/L, heterotrophic respiration was 207 mg/L, nitrification respiration was 197 mg/L, test on activated sewage sludge microorganisms, OECD 209 (2010) Timmer, N., 2020.

Key value for chemical safety assessment

EC50 for microorganisms:
367 mg/L
EC10 or NOEC for microorganisms:
134 mg/L

Additional information

The objective of the study was to evaluate the test item for its ability to adversely affect aerobic microbial treatment plants and, if possible, to determine the EC50 and/or the no - observed effect concentration (NOEC).A final test was performed preceded by a combined limit/range-finding test.The study met the validity criteria prescribed by the study plan and was considered to be valid.


The test was conducted on the degradation products as the hydrolysis half-life of the test item was found to be < 1 hour.This was acheived by stirring the parent prior to microorganism exposure, for 3 hours (i.e. 6 times the half life). The time was chosen based on preliminary data showing that 3 hours is equal to or greater than 6 degradation half-lives of the test item, which is generally sufficient to ensure that the test solution contains only degradation products. This was supported by the hydrolysis study (OECD 111), which showed that hydrolysis at pH 7 and 9 was so rapid that it was not possible to measure the half lives, where measures were taken at least every 10 minutes (Determination of Physico-chemical Properties of N-Phenyl-diethanolamine, reaction products with formaldehyde, Petrovic, D, Study No. 20194879, 2020).  The OECD 209 was conducted at pH 7.0 to 8.4.  Further support for the fact only degradation products were tested comes from the OECD 201, 202 and 203 studies which showed the parent was completely degraded after 3 hours of stirring in environmentally relevant medium and that the concentration of the transformation/degradation products remained within 80-120 % of the concentration at time 0 hours of the experiments (OECD 201 Augusiak, J.A., 2020; OECD 202 Augusiak, J.A., 2020; OECD 203 Augusiak, J.A., 2020).


This is in accordance with OECD document No. 23 (Second edition, 2019) and ECHA R.7b (v4.0, 2017).


 


The experiment was conducted with and without ATU in order to derive effects on the inhibition of total and heterotrophic respiration and nitrification. The experimental set up followed the OECD 209 guideline without deviation. Tested concentrations were 0, 180, 320, 560 and 1000 mg/L.


 


ECx and NOEC values were:


NOEC total respiration: 180 mg/L


NOEC heterotrophic respiration: 180 mg/L


NOEC nitrification: 320 mg/L


EC50 for total respiration was 529 mg/L (95% confidence interval: 483 - 578 mg/L).


EC50 for heterotrophic respiration was 602 mg/L (95% confidence interval: 422 - 850 mg/L).


EC50 for nitrification respiration was 367 mg/L (95% confidence interval: 118 - 1141 mg/L).


The test substance inhibited heterotrophic respiration and nitrification, the lowest EC10 was 134 mg/L for total respiration.

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